Last Updated: December 4, 2024
Introduction to Amino Acid Metabolism
All tissues have some capability for synthesis of the non-essential amino acids, amino acid remodeling, and conversion of non-amino acid carbon skeletons into amino acids and other derivatives that contain nitrogen. However, the liver is the major site of nitrogen metabolism in the body. In times of dietary surplus, the potentially toxic nitrogen of amino acids is eliminated via transaminations, deamination, and urea formation; the carbon skeletons are generally conserved as carbohydrate, via gluconeogenesis, or as fatty acid via fatty acid synthesis pathways. In this respect amino acids fall into three categories: glucogenic, ketogenic, or glucogenic and ketogenic. Glucogenic amino acids are those that give rise to a net production of pyruvate or TCA cycle intermediates, such as 2-oxoglutarate (α-ketoglutarate) or oxaloacetate, all of which are precursors to glucose via gluconeogenesis. All amino acids except lysine and leucine are at least partly glucogenic. Lysine and leucine are the only amino acids that are solely ketogenic, giving rise only to acetyl-CoA or acetoacetyl-CoA, neither of which can bring about net glucose production.
A small group of amino acids comprised of isoleucine, phenylalanine, threonine, tryptophan, and tyrosine give rise to both glucose and fatty acid precursors and are thus, characterized as being glucogenic and ketogenic. Finally, it should be recognized that amino acids have a third possible fate. During times of starvation the reduced carbon skeleton is used for energy production, with the result that it is oxidized to CO2 and H2O.
Intestinal Amino Acid Uptake
Digestion of Dietary Proteins
Dietary amino acids can be consumed as free amino acids or, more often, they are acquired from digested dietary proteins that are hydrolyzed through the concerted actions of gastric and pancreatic peptidases. Dietary protein digestion begins in the stomach via the actions of the pepsins, and continues within the lumen of the duodenum. Within the small intestine there are two principal pancreatic enzymes involved in protein digestion; trypsin and chymotrypsin. Several additional pancreatic peptidases play a lesser role in peptide digestion and include the carboxypeptidases and the elastases.
The initial enzymes involved in protein digestion are the gastric pepsins. Pepsins are derived from the precursor zymogen, pepsinogen. Pepsinogen is released from stomach chief cells. Pepsins are released from pepsinogen via acid-induced autocatalysis. Pepsins hydrolyze peptide bonds on the C-terminal side of aromatic and hydrophobic amino acids. Approximately 20% of overall protein digestion is accomplished via the actions of pepsins. Due to the acidic pH optimum for the action of pepsins, these enzymes are inhibited when the gastric juice (chyme) passes from the stomach and is mixed with alkaline pancreatic juice in the duodenum.
The remainder of protein digestion occurs within the duodenum and jejunum of the small intestine. Digestion here is primarily the result of the actions of the pancreatic trypsins and the pancreatic chymotrypsins, and to a lesser extent by the pancreatic elastases, and by the pancreatic carboxypeptidases A1 and A2.
Humans express three genes that encode members of the trypsin family of serine proteases. These genes are identified as protease, serine 1, 2, and 3 (encoded by the PRSS1, PRSS2, and PRSS3 genes, respectively). Expression of the PRSS1 gene is exclusive to the pancreas while expression of the PRSS2 and PRSS3 genes is at highest levels in the pancreas and at very low levels in the intestines.
The PRSS1 and PRSS2 genes are located in a cluster on chromosome 7q34. The PRSS1 gene is composed of 5 exons that encode a 247 amino acid preproprotein.
The PRSS2 gene is composed of 6 exons that generate two alternatively spliced mRNAs that encode a 261 amino acid preproprotein (isoform 1) and a 247 amino acid preproprotein (isoform 2).
The PRSS3 gene is located on chromosome 9p13.3 and is composed of 8 exons that generate four alternatively spliced mRNAs, each of which encode a distinct preproprotein isoform.
Humans express two genes encoding chymotrypsinogen identified as CTRB1 and CTRB2 with the CTRB1 encoded enzyme being the predominant pancreatic chymotrypsinogen. Expression of both the CTRB1 and CTRB2 genes is exclusive to the pancreas.
The CTRB1 and CTRB2 genes are associated in a head-to-tail orientation on chromosome 16q23.1 and both are composed of 7 exons. The CTRB1 gene generates 2 alternatively spliced mRNAs that encode a 263 amino acid precursor protein (isoform 1) and a 233 amino acid precursor protein (isoform 2). The CTRB2 gene encodes a 263 amino acid precursor protein.
Humans express a large family of 25 genes that encode enzymes of the carboxypeptidase family. The majority of these enzymes are involved in protein processing and are not involved in digestive processes. The term carboxypeptidase A refers to the fact that these enzymes hydrolyze peptide bonds where the amino acid on the C-terminal side of the bond is an aromatic or aliphatic amino acid. The carboxypeptidase A family includes six genes where the CPA1 and CPA2 genes encode pancreatic digestive enzymes.
Humans express eight genes that encode enzymes of the elastase family with the chymotrypsin-like elastase family members (excluding the CELA1 encoded enzyme) being pancreatic digestive enzymes. The digestive elastases are chymotrypsin-like elastase 2A, 2B, 3A, and 3B (CELA2A, CELA2B, CELA3A, and CELA3B genes, respectively).
Active trypsin is generated via the action of enteropeptidase on pancreatic trypsinogen. Enteropeptidase is an enzyme secreted by intestinal epithelial cells of the crypts of Lieberkühn and the enzyme resides in the brush-border (apical) membranes of duodenal mucosal (enterocytes) cells. The generated trypsin then cleaves more trypsinogen to trypsin, as well as chymotrypsinogens, proelastases, and procarboxypeptidases to their active forms.
Enteropeptidase is encoded by the TMPRSS15 (transmembrane serine protease 15) gene. The TMPRSS15 gene is located on chromosome 21q21.1 and is composed of 30 exons that encode a 1019 amino acid precursor protein.
Following digestion, free amino acids, as well as peptides (2–6 amino acids in length) are absorbed by enterocytes of the proximal jejunum. Some absorption also occurs in the duodenum and a minor amount in the ileum. Although there is little nutritional significance to whole protein absorption, some undigested dietary protein does get absorbed by colonic (large intestine) enterocytes.
Amino Acid and Peptide Transporters
The absorption of most amino acids from the intestine requires an active transport process that is dependent upon Na+ or H+ co-transport. There are numerous amino acid transporters encompassing at least six major families of transport systems which are sometimes grouped into three broad categories. These three broad categories are the neutral amino acid (monoamino monocarboxylic) transporters, the dibasic (and cysteine) amino acid transporters, and the acidic (dicarboxylic) amino acid transporters.
Amino acid transporters have numerous different designations based upon both historical characterizations and newer standardized nomenclatures. The initial classification of amino acid transporters reflected a systems-based approach to their classification. Within the context of amino acid transporter nomenclature the use of lowercase acronyms indicates Na+-independent transporters (e.g. b0,+ and asc), whereas the use of uppercase acronyms indicates Na+-dependent transporters (e.g. B0,+ and ASC). All of the amino acid transporters are members of the solute carrier (SLC) family of transporters and as such the genes encoding these transporters all use the SLC nomenclature.
The amino acid transporters involved in intestinal amino acid uptake are members of the SLC1, SLC6, SLC7, SLC36, SLC38, and SLC43 gene families. The transport of amino acids occurs in all other cells and so these transport systems are expressed throughout the body. They are critically important within the context of the brain and the kidney. Many of the transporters that are required for intestinal amino acid uptake are also expressed in the proximal tubules of the kidneys where they are responsible for reabsorption of amino acids from the glomerular filtrate.
Due to the inconsistent usage of the varied nomenclature systems for amino acid transporters, a nomenclature system encompassing five transport activities has been proposed. These five system designations are: 1) the neutral system (or methionine preferring system) which transports all neutral amino acids; 2) the basic system which is responsible for the transport of cationic amino acids in addition to cystine; 3) the acidic system which transports glutamate and aspartate; 4) the iminoglycine system which transports proline, hydroxyproline, and glycine; and 5) the β-amino acid system which transports taurine and β-alanine.
Neutral Amino Acid Transporters
The neutral amino acid transporters were originally subdivided into several subfamilies with all but one family being Na+-dependent transporters. These original designations were the system A, ASC, N, beta (β), GLY, IMINO, PHE, B0, and L transporters that are Na+-dependent and the asc transporter which is Na+-independent. The B of the system B0 transporters refers to “broad” whereas the superscript “0” of the system refers to neutral amino acid transport.
The system A and system B0 transporters were those defined by their preference for alanine and other small and polar neutral amino acids. The A family transporters are also referred to as Na+-dependent neutral amino acid transporters (SNAT) and they are encoded by the SLC38A2 (SNAT2) and SLC38A4 (SNAT4) genes. The B0 family transporters are encoded by the SLC6A15 (B0AT2; B0-type Amino acid Transporter 2) and SLC6A19 (B0AT1) genes, respectively. The B0AT1 transporter requires an accessory protein within the renal epithelial cell apical membrane as well as one in the intestinal enterocyte apical membrane. The renal accessory protein is collectrin and the intestinal accessory protein is angiotensin converting enzyme 2 (ACE2). B0AT1 transports all neutral amino acids but prefers branched-chain amino acids and methionine.
The Na+-dependent system ASC transporters (ASCT) were defined by their preference for Alanine, Serine, and Cysteine, hence the ASC nomenclature. The ASCT1 transporter is encoded by the SLC1A4 gene and the ASCT2 transporter is encoded by the SLC1A5 gene.
The system L amino acid transporters (LAT), which are Na+-independent, were defined by their preference for leucine and other large (bulky) hydrophobic neutral amino acids. There are two subfamilies of system L transporters, one which is monomeric and the other which in heterodimeric. The monomeric LAT are identified as LAT3 and LAT4 and they are encoded by the SLC43A1 and SLC43A2 genes, respectively. The heterodimeric LAT transporters are described below.
The system N amino acid transporters (SNAT) show preference for glutamine, asparagine, and histidine and are encoded by the SLC38A3 (SNAT3) and SLC38A5 (SNAT5) genes.
The β system transporter is encoded by the SLC6A6 gene and transports β-alanine (3-aminopropanoic acid) and taurine. Beta-alanine is a naturally occurring β-amino acid where the amino group is attached to the β-carbon (two carbons from the carboxylic acid group) instead of the α-carbon. A primary source of β-alanine is the catabolism of the pyrimidines, uracil and cytosine.
The IMINO transporter is encoded by the SLC6A20 gene and transports proline and hydroxyproline.
The GLY system is the glycine transporter encoded by the SLC6A18 gene.
The asc system transporter is one of the heterodimeric transporters described below.
Acidic Amino Acid Transporters
The acidic amino acid transporters were also subdivided into several subfamilies, all of which are Na+-dependent transporters. The acidic transporter subfamilies include the system B0,+, b0,+, y+, y+L, XAG–, and Xc– transporters. Within this nomenclature a “y” and superscript “+” refers to cationic transporters, an “x” and a superscript “-” refers to anionic transporters, and a “z” refers to neutral transporters.
The B0,+ transporter is encoded by the SLC6A14.
The b0,+ transporter is one of the heterodimeric transporters described in the next section.
The y+ transporter is encoded by the SLC7A1 gene.
The y+L transporter is one of the heterodimeric transporters described in the next section.
The XAG– transporters are potassium-dependent glutamate and aspartate transporters. The XAG– transporters (SLC1 family) are now more commonly called the excitatory amino acid transporters (EAAT) with there being five members EAAT1 (SLC1A3), EAAT2 (SLC1A2), EAAT3, (SLC1A1), EAAT4 (SLC1A6), and EAAT5 (SLC1A7).
The Xc– transporters are heterodimeric cystine and glutamate antiporters that transport cystine in the opposite direction to that of glutamate.
Heterodimeric Amino Acid Transporters
The transporters of the asc, L, b0,+, y+L, and Xc– subfamilies are all heterodimeric transporters. These heterodimeric transporters are composed of a light chain and a heavy chain. In all but the case of the b0,+ transporter the heavy chain subunit is the SLC3A2 encoded protein identified as 4F2hc (4F2 cell-surface antigen heavy chain). The 4F2 heterodimer is also known by the designation CD98.
The asc transporter is composed of a light chain (ascAT1) encoded by the SLC7A10 gene and the 4F2hc heavy chain.
The L type transporter subfamily is composed of two members that are derived from two different light chain subunits associated with the 4F2hc heavy chain. The two L type transporter light chains are encoded by the SLC7A5 (LAT1) and SLC7A8 (LAT2) genes.
The b0,+ transporter is composed of the SLC7A9 encoded light chain (b0,+AT1) and the heavy chain identified as rBAT encoded by the SLC3A1 gene.
The y+L transporter subfamily is composed of two members that are derived from two different light chain subunits associated with the 4F2hc heavy chain. The two y+L light chain subunits are encoded by the SLC7A6 (y+LAT2) and SLC7A7 (y+LAT1) genes.
The Xc– transporter is composed of the SLC7A11 (xCT) encoded light chain and the 4F2hc heavy chain.
Intestinal Peptide Transport
Intestinal uptake of peptides, primarily dipeptides and tripeptides, involves H+ co-transporters which are also members of the SLC family. The most abundant peptide transporter is PepT1 (encoded by the SLC15A1 gene) but there are three additional peptide transporters within the SLC15 subfamily.
Within intestinal enterocytes the absorbed peptides are hydrolyzed to free amino acids via enterocyte cytoplasmic peptidases. The free amino acids are then transported across the basolateral membranes of enterocytes where they enter the superior mesenteric vein which feeds into the portal vein entering the liver.
Primary Amino Acid Transporters of the Intestines
Among the many different amino acids transporters from the different families described above, those that are responsible for intestinal uptake are indicated in the following Table.
Table of Major Intestinal Amino Acid Transporters
| Gene(s) | Transporter | Functions / Comments |
| SLC1A1 | EAAT3 | member of the Excitatory Amino Acid Transporter family; originally designated the XAG– transporters which are K+-dependent glutamate and aspartate transporters; EAAT3 transports aspartate, glutamate, and cysteine; co-transports Na+ into and K+ out of cells; expressed in small intestine, kidney, brain, and liver |
| SLC1A5 | ASCT2 | Alanine/Serine/Cysteine Transporter 2; in addition ASCT2 transports threonine, glutamine, and asparagine; is an antiporter that transports amino acids into cells in exchange for Na+ transport out of the cell; expressed in most tissues |
| SLC3A2/SLC7A6 | y+LAT2/4F2hc | is one of the heterodimeric amino acid transporters; y+ refers to cationic, LAT2 is Large and neutral Amino acid Transporter 2; transports methionine, lysine, isoleucine, leucine, cysteine, histidine, arginine, and glutamine; is an antiporter that exchanges amino acids for Na+; expressed in most tissues; primary transporter for arginine efflux from cells |
| SLC3A2/SLC7A7 | y+LAT1/4F2hc | is one of the heterodimeric amino acid transporters; y+ refers to cationic, LAT1 is Large and neutral Amino acid Transporter 1; 4Fhc is the heavy chain; transports methionine, lysine, isoleucine, leucine, cysteine, histidine, arginine, and glutamine; is an antiporter that exchanges amino acids for Na+; expressed in small intestine, lungs, kidneys, and blood |
| SLC3A2/SLC7A8 | LAT2/4F2hc | is one of the heterodimeric amino acid transporters; LAT2 is Large and neutral Amino acid Transporter 2; 4F2hc is the heavy chain; transports all neutral amino acids except proline; expressed in most tissues |
| SLC3A1/SLC7A9 | rBAT/b0,+AT | is one of the heterodimeric amino acid transporters; b0,+ refers to broad and cationic, AT refers to Amino acid Transporter; rBAT is the heavy chain and b0,+AT is the light chain; transports lysine, arginine, ornithine, and cystine; expressed in small intestines and kidneys |
| SLC6A6 | TauT | Taurine Transporter; taurine and β-alanine transporter; co-transports Na+ or Cl– ions; expressed in most tissues |
| SLC6A9 | GlyT1 | glycine transporter; co-transports Na+ or Cl– ions; expressed in most tissues |
| SLC6A14 | ATB0,+ | Amino acid Transporter Broad (0) and cationic (+); transports neutral and cationic amino acids as well as β-alanine; co-transports Na+ or Cl– ions; expressed in large intestines and lungs |
| SLC6A19 | B0AT1 | transport all neutral amino acids; the required accessory protein, ACE2, cleaves peptides into free amino acids that can then be transported; co-transports Na+ ions; expressed in small intestines and kidneys |
| SLC6A20 | SIT1 | System IMINO Transporter 1; transports proline and hydroxyproline; co-transports Na+ or Cl– ions; expressed in small intestines, kidneys, and brain |
| SLC16A10 | TAT1 | T-type Amino acid Transporter 1; transports phenylalanine, tyrosine, and tryptophan; expressed at low levels in small intestine, predominant expression is in skeletal muscle and pancreas |
| SLC36A1 | PAT1 | Proton Amino acid Transporter 1; also known as imino acid carrier; transports proline, glycine, alanine, β-alanine, and the neurotransmitter GABA; expressed in small and large intestines, brain, and blood |
| SLC38A2 | SNAT2 | Small Neutral Amino acid Transporter 2; transports glycine, alanine, serine, leucine, glutamine, methionine, cysteine, histidine, asparagine, and proline; co-transports Na+ ions; expressed in numerous tissues |
| SLC38A3 | SNAT3 | Small Neutral Amino acid Transporter 3; transports asparagine, histidine, and glutamine; co-transports Na+ or H+ ions; expressed predominantly in kidneys and liver |
| SLC38A4 | SNAT4 | Small Neutral Amino acid Transporter 4; transports alanine, glycine, serine, methionine, asparagine, cysteine, and glutamine; co-transports Na+ ions; expressed predominantly in the liver |
| SLC38A5 | SNAT5 | Small Neutral Amino acid Transporter 5; transports glycine, serine, histidine, asparagine, and glutamine; co-transports Na+ or H+ ions; expressed in numerous tissues |
| SCL43A2 | LAT4 | Large and neutral Amino acid Transporter 4; transports methionine, phenylalanine, leucine, and isoleucine; expressed in small intestine, kidneys, skeletal muscle, lung, and blood |
Essential vs. Nonessential Amino Acids
| Nonessential | Alanine, Asparagine, Aspartate, Cysteine, Glutamate, Glutamine, Glycine, Proline, Serine, Tyrosine |
| Essential | Arginine*, Histidine, Isoleucine, Leucine, Lysine, Methionine*, Phenylalanine*, Threonine, Tryptophan, Valine |
*The amino acids arginine, methionine and phenylalanine are considered essential for reasons not directly related to lack of synthesis. Arginine is synthesized by mammalian cells but at a rate that is insufficient to meet the growth needs of the body and the majority that is synthesized is cleaved to form urea. Methionine is required in large amounts to produce cysteine if the latter amino acid is not adequately supplied in the diet. Similarly, phenylalanine is needed in large amounts to form tyrosine if the latter is not adequately supplied in the diet.
Glutamate Biosynthesis
Glutamate can be synthesized by two distinctly different reaction pathways. In one reaction glutamate is synthesized by the reductive amination of 2-oxoglutarate (α-ketoglutarate) catalyzed by glutamate dehydrogenase (GDH); it is thus a nitrogen-incorporating reaction. In the second type of reaction glutamate is formed from 2-oxoglutarate by aminotransferase reactions, with the amino nitrogen being donated by a number of different amino acids. Thus, glutamate is a general collector of amino nitrogen, a critical reaction in overall nitrogen homeostasis as discussed in detail in the Nitrogen Metabolism and the Urea Cycle page.
Humans express two distinct glutamate dehydrogenase genes identified as GLUD1 and GLUD2.
The GLUD1 gene encoded enzyme is the primary glutamate dehydrogenase (GDH1) activity in most tissues. This enzyme is localized to the mitochondrial matrix and functions as a homohexameric complex. The GLUD1 gene is located on chromosome 10q23.2 and is composed of 18 exons that generate seven alternatively spliced mRNAs that collectively encode three distinct protein isoforms.
The GLUD2 gene is thought to have arisen as a result of a retrotranspostitional event to the X chromosome. The GLUD2 gene is located at Xq24 and is an intronless gene encoding a precursor protein of 558 amino acids. The GLUD2 encoded protein (GDH2) also forms a homohexameric complex in the mitochondrial matrix. Expression of the GLUD2 gene is highest in neural tissues and the regulatory controls over this form of the enzyme are distinct from the GLUD1 encoded enzyme. The GDH2 complex is not inhibited by GTP which allows astrocyte GDH2 to continue to function under conditions of intense excitatory neurotransmission allowing these cells to handle the increased loads of the neurotransmitter glutamate.
Glutamate can also be derived from glutamine via the action of glutaminase. Indeed, the interconversion of glutamine and glutamate represents a critical pathway in overall nitrogen, acid-base, and energy homeostasis in the body as described in greater detail below. Glutaminase activity is present in many tissues such as the liver, kidney, small intestine, and neurons. The primary subcellular site of glutaminase localization is the mitochondria.
There are two distinct glutaminase genes in humans identified as GLS (encoding the GLS1 enzyme) and GLS2 (encoding the GLS2 enzyme).
The GLS gene is located on chromosome 2q32.2 and is composed of 22 exons that undergo alternative splicing to yield two mRNAs generating two isoforms of the enzyme. These two GLS-derived isoforms are often referred to as glutaminase C (GAC) and kidney-type glutaminase (KGA) but are collectively the glutaminase 1 (GLS1) enzymes. The GLS encoded isoforms of glutaminase are primarily expressed in the kidneys. GLS encoded kidney-type glutaminase is a protein of 669 amino acids and the GLS encoded glutaminase C is a protein of 598 amino acids.
The GLS2 gene encoded glutaminase was originally thought to be liver specific but is in fact expressed in numerous tissues and is important in the glutamate-glutamine cycle in the brain. The GLS2 encoded glutaminase was originally characterized as dependent on inorganic phosphate (Pi) for activity and is, therefore, also referred to as phosphate-activated glutaminase, PAG.
The GLS2 gene is located on chromosome 12q13.3 and is composed of 19 exons that undergo alternative splicing to yield four mRNAs. The two major GLS2-derived mRNAs have been identified as LGA and GAB. The GAB mRNA contains all of the coding exons and the LGA mRNA lacks exon 1. The LGA mRNA results from the use of an alternative promoter present in intron 1 of the GLS2 gene.
As a result of the use of alternative promoters and through alternative splicing there are four major isoforms of glutaminase enzymes. These four isoforms exhibit different molecular, kinetic, protein interacting partners, and regulatory properties. As indicated above, one of the first characterized differences was the dependence on Pi for activity. Both the GLS gene encoded enzymes and the GLS2 encoded enzymes require phosphate for activity. With GLS gene derived enzymes the GAC isoform is the most responsive to phosphate. This difference in sensitivity is believed to be the result of the differences in the C-terminus of the KGA and GAC isoforms encoded by the GLS gene. The GLS encoded enzymes are inhibited by glutamate but the GLS2 encoded enzyme is not. The GLS2 encoded enzyme is activated by ammonia but the GLS encoded enzymes are not.
Glutamine Biosynthesis
Glutamine is synthesized from glutamate via the action of glutamine synthetase. The glutamine synthetase enzyme is encoded by the glutamate-ammonia ligase (GLUL) gene. The GLUL gene is located on chromosome 1q25.3 and is composed of 9 exons that generate three alternatively spliced mRNAs, each of which encode the same 373 amino acid protein.
Ultimately, however, glutamine is derived from 2-oxoglutarate (α-ketoglutarate) via the sequential actions of amino transferases, or glutamate dehydrogenase, that yield glutamate from 2-oxoglutarate and then via the glutamine synthetase reaction acting on glutamate.
Glutamine Metabolism in Growth and Cancer
Glutamine represents the most abundant amino acid in the blood with levels that are in the range of 500μM. This level of glutamine accounts for over 20% of the total pool of free amino acids in the blood. Although briefly outlined here, the details of the significance of glutamine metabolism in cancer are covered in the Metabolic Alterations Associated with Cancer page.
Glutamine is also critical in the biosynthesis of nucleotides for RNA and DNA synthesis as is required in rapidly proliferating cells and cancer cells. Glutamine can serve as a precursor for aspartate through the concerted actions of glutaminase, yielding glutamate, followed by transfer of the nitrogen from glutamate to oxaloacetic acid (OAA) forming aspartate. The oxaloacetic acid can also be derived from glutamine, thus indicating this pivotal role of glutamine in overall nucleotide homeostasis. The aspartate that can be derived from glutamine is required as a source of carbon for both purine and pyrimidine nucleotide synthesis.
In cancer cells, the expression of enzymes involved in glutamine metabolism varies widely and is related to factors such as tissue of origin and the overall genotype of the specific cancer. As indicated in the previous section, humans express two genes encoding glutaminase enzymes, GLS encoding the GLS1 enzyme and GLS2 encoding the GLS2 enzyme. The GLS gene is more widely expressed and is the glutaminase encoding gene believed to be of more significance in cancer cell metabolism.
Glutamate dehydrogenase activity is also altered in cancer cells. Glutamate dehydrogenase is allosterically activated by leucine, the levels of which will be higher in cancer cells due to increased activity of various amino acid transporters. In the direction of glutamate deamination, glutamate dehydrogenase is activated by ADP. Therefore, low cellular energy states will lead to induction of 2-oxoglutarate production via the glutamate dehydrogenase reaction allowing for increased ATP synthesis from the TCA cycle-derived NADH and FADH2.
Aspartate Biosynthesis
Aspartate can be formed in a transamination reaction or via the deamination of asparagine. The transamination reaction is catalyzed by aspartate transaminase, AST. This reaction uses the α-keto acid, oxaloacetate, as the amino acceptor and glutamate as the primary amino group donor.
Humans express two AST enzymes, one that is localized in the cytosol and the other is localized to the mitochondria.
The cytosolic AST enzyme is encoded by the glutamate oxaloacetic transaminase 1 gene (GOT1). The GOT1 gene is located on chromosome 10q24.2 and is composed of 9 exons that encode a 413 amino acid protein.
The mitochondrial AST enzyme is encoded by the GOT2 gene which is located on chromosome 16q21 and is composed of 10 exons that generate two alternatively spliced mRNAs encoding precursor proteins of 430 amino acids (isoform 1) and 387 amino acids (isoform 2).
Aspartate, formed by deamination of asparagine, is catalyzed by asparaginase. Humans express at least three enzymes that can catalyze the deamination of asparagine to aspartate. However, the true primary physiologic substrates for these enzymes may not be asparagine.
One enzyme was originally identified as a lysophospholipase due to its ability to hydrolyze lysophospholipids in addition to it being subsequently shown to harbor asparaginase activity. This enzyme is encoded by the asparaginase (ASPG) gene. The ASPG gene is located on chromosome 14q32.33 and is composed of 20 exons that generate two alternatively spliced mRNAs encoding proteins of 573 amino acids (isoform 1) and 555 amino acids (isoform 2).
A second human enzyme with asparaginase activity is the enzyme N-aspartyl-β-glucosaminidase (also called aspartylglucosaminidase) which is encoded by the AGA gene. The AGA gene is located on chromosome 4q34.3 and is composed of 9 exons that generate two alternatively spliced mRNAs. These mRNAs encode a 346 amino acid protein (isoform 1) and a 336 amino acid protein (isoform 2). The 346 amino acid protein exhibits an approximate mass of 35 kDa. Following synthesis the protein is cleaved to generate two subunits. The α-subunit is 27 kDa and the β-subunit is 17 kDa and these subunits form a heterotetrameric enzyme. The AGA encoded protein has as its primary function the hydrolysis of carbohydrates linked to asparagine residues in glycoproteins. Defects in the AGA gene result in aspartylglucoasminuria which is a lysosomal storage disease.
The third human enzyme with asparaginase activity is called asparaginase like 1 (also called asparaginase and isoaspartyl peptidase 1) which is encoded by the ASRGL1 gene. In addition to being able to hydrolyze asparagine to aspartate, the ASRGL1 encoded enzyme hydrolyzes isoaspartyl peptide linkages. The ASRGL1 gene is located on chromosome 11q12.3 and is composed of 10 exons that generate two alternatively spliced mRNAs that express the same 308 amino acid protein.
Asparaginases from bacteria have significant clinical utility as they are used in the treatment of several forms of hematopoietic malignancies that express low levels of the ASNS gene and, therefore, rely on extracellular asparagine for their rapid growth. Leukemias that are asparaginase resistant have elevated levels of expression of the ASNS gene. Also, cancers that are capable of synthesizing asparagine de novo via asparagine synthetase are less responsive to
asparaginase therapy.
Asparagine Biosynthesis
Asparagine is synthesized from aspartate via an amidotransferase reaction catalyzed by asparagine synthetase. Asparagine synthetase is encoded by the ASNS gene. The ASNS gene is located on chromosome 7q21.3 and is composed of 16 exons that generate seven alternatively spliced mRNAs that collectively encode three protein isoforms.
Clinical significance of asparagine synthesis is demonstrated by the fact that the level of expression of the ASNS gene is correlated to metastatic potential in numerous forms of cancer. Additionally, treatment of several cancer types with the enzyme asparaginase, or dietary restriction of asparagine, reduces metastasis but does not alter the growth of primary tumors. Conversely, enforced expression of the ASNS gene strongly promotes metastatic potential.
Alanine Biosynthesis and the Glucose-Alanine Cycle
Aside from its role in protein synthesis, alanine is second only to glutamine in prominence as a circulating amino acid. In this capacity it serves a unique role in the transfer of nitrogen from peripheral tissue to the liver. Alanine is transferred to the circulation by many tissues, but mainly by muscle, in which alanine is formed from pyruvate at a rate proportional to intracellular pyruvate levels. Liver accumulates plasma alanine, reverses the transamination that occurs in muscle, and proportionately increases urea production. The pyruvate is either oxidized or converted to glucose via gluconeogenesis. When alanine transfer from muscle to liver is coupled with glucose transport from liver back to muscle, the process is known as the glucose-alanine cycle. The key feature of the cycle is that in 1 molecule, alanine, peripheral tissue exports pyruvate and ammonia (which are potentially rate-limiting for metabolism) to the liver, where the carbon skeleton is recycled and most nitrogen eliminated.
There are two main pathways to production of muscle alanine: directly from protein degradation, and via the transamination of pyruvate by alanine transaminase, ALT (historically referred to as serum glutamate-pyruvate transaminase, SGPT).
Humans express two genes encoding alanine transaminase. These genes are identified as GPT (also designated GPT1) and GPT2. The enzyme encoded by the GPT gene is localized to the cytosol, whereas the GPT2 encoded enzyme is localized to the mitochondria.
The GPT gene is located on chromosome 8q24.3 and is composed of 12 exons that generate three alternatively spliced mRNAs, all of which encode the same 496 amino acid protein.
The GPT2 gene is located on chromosome 16q11.2 and is composed of 13 exons that generate two alternatively spliced mRNAs that encode proteins of 523 amino acids (isoform 1) and 423 amino acids (isoform 2).
Cysteine, Methionine, and S-Adenosylmethionine Biosynthesis
The essential amino acid methionine plays many crucial roles in the body, most of which are not related to its function in the synthesis of proteins. In the context of the role of methionine in protein synthesis it also serves as the precursor for the synthesis of the amino acid cysteine. The sulfur for cysteine synthesis comes from methionine through a series of interrelated reaction pathways. In addition to its role in the synthesis of cysteine, methionine is critical in the synthesis of the methyl donor, S-adenosylmethionine (SAM, SAMe, or AdoMet).
Synthesis of S-Adenosylmethionine
In the synthesis of S-adenosylmethionine (SAM), methionine and the adenosine from ATP are condensed via the action of methionine adenosyltransferase (MAT). In the production of SAM all phosphates of an ATP are lost: one as Pi and two as PPi, which itself is converted to two moles of Pi via the action of either of two enzymes (PPA1 or PPA2) of the pyrophosphatase (inorganic) family. In the MAT catalyzed reaction it is adenosine which is transferred to methionine and not AMP. MAT is also called S-adenosylmethionine synthetase.
There are three MAT genes in humans identified as MAT1A, MAT2A, and MAT2B. The MAT enzymes encoded by the MAT1A gene function as either a homotetramer identified as MAT I (called the alpha form), or as a homodimer identified as MAT III (called the beta form). The MAT I and MAT III isoforms are only expressed in the liver. The MAT enzyme encoded by the MAT2A gene is identified as MAT II (called the gamma form). The MAT II enzyme is expressed in several non-hepatic tissues. The MAT2B encoded protein is the non-catalytic regulatory subunit.
The MAT1A gene is located on chromosome 10q22.3 and is composed of 9 exons that encode a protein of 395 amino acids. Expression of the MAT1A gene is very nearly exclusive to the liver.
The MAT2A gene is located on chromosome 2p11.2 and is composed of 9 exons that encode a protein of 395 amino acids. Expression of the MAT2A gene is ubiquitous.
The MAT2B gene is located on chromosome 5q34 and is composed of 8 exons that generate two alternatively spliced mRNAs encoding the MAT 2 beta isoform 1 (334 amino acids) and the MAT 2 beta isoform 2 (323 amino acids) enzymes. Expression of the MAT2B gene is ubiquitous.
Mutations in the MAT1A gene are associated with the disorder referred to as methionine adenosyltransferase I/III deficiency. This disorder can be inherited as an autosomal recessive disease or as an autosomal dominant disease. In most individuals the disorder is associated with a benign phenotype, with pathology only seen in patients with plasma methionine levels exceeding 800μM. Symptoms in these patients result from demyelination in the CNS.
SAM serves as a precursor for numerous different methyl transfer reactions where one of the most physiologically significant reactions is the conversion of norepinephrine to epinephrine (see Amino Acid Derivatives: Neurotransmitters, Nitric Oxide, and More page). The result of methyl transfer is the conversion of SAM to S-adenosylhomocysteine (SAH). S-adenosylhomocysteine is then cleaved by adenosylhomocyteinase (also called S-adenosylhomocysteine hydrolase) to yield homocysteine and adenosine.
Adenosylhomocysteinase is encoded by the AHCY gene. The AHCY gene is located on chromosome 20q11.22 and is composed of 17 exons that generate six alternatively spliced mRNAs encoding three distinct proteins isoforms of 432 amino acids (isoform 1), 404 amino acids (isoform 2), and 434 amino acids (isoform 3).
Transmethylation reactions employing SAM are extremely important, but in the context of cysteine biosynthesis, the role of S-adenosylmethionine in transmethylation is secondary to the production of homocysteine (essentially a by-product of transmethylase activity).
Methionine Synthesis from Homocysteine
The primary pathway for the conversion of homocysteine back to methionine is catalyzed by methionine synthase (also called homocysteine methyltransferase). This reaction was also discussed in the context of vitamin B12-requiring enzymes in the Vitamins: Water and Fat Soluble page. The methionine synthase reaction represents the clinically most significant of only two vitamin B12-requiring enzymes in human cells. Methionine synthase, in addition to B12, requires the folate-derived vitamin co-factor, N5-methyltetrahydrofolate (N5-methylTHF; or simply methylTHF).
Methionine synthase is encoded by the MTR gene (5-methyltetrahydrofolate-homocysteine methyltransferase). The MTR gene is located on chromosome 1q43 and is composed of 33 exons that generate four alternatively spliced mRNAs, each of which encode a distinct protein isoform.
Mutations in the MTR gene are a rare cause of homocysteinemia/homocystinuria.
During the conversion of homocysteine to methionine the cobalt within the methyl-cobalamin cofactor required of methionine synthase can become oxidized. The cobalt of cobalamin can exist in three oxidation states, +1 [cob(I)alamin], +2 [cob(II)alamin], and +3 [cob(III)alamin]. Within the context of vitamin B12 function the cobalamin is in the +1 or +2 state. The oxidation of cobalt within the context of the methionine synthase reaction converts cob(I)alamin to cob(II)alamin. The reduction of the cob(II)alamin state to the cob(I)alamin state is required to allow methionine synthase to continue to convert homocysteine to methionine.
The reduction of cobalt in cobalamin of methionine synthase is catalyzed by methionine synthase reductase. Methionine synthase reductase is encoded by the MTRR (5-methyltetrahydrofolate-homocysteine methyltransferase reductase) gene. The MTRR gene is located on chromosome 5p15.31 and is composed of 21 exons that generate five alternatively spliced mRNAs, each of which encode the same 698 amino acid protein.
Mutations in the MTRR gene are a rare cause of homocysteinemia/homocystinuria.
Role of Betaine in Methionine Synthesis
Homocysteine can be converted back to methionine via a folate-independent pathway involving the enzyme betaine-homocysteine S-methyltransferase, BHMT. Betaine (also called trimethylglycine) is an abundant amino acid that, although it is not found in proteins, play a critical biochemical roles in humans. The majority of betaine is obtained from the diet and can also be synthesized from choline.
In order to synthesize betaine, choline is first transported into the mitochondria via the action of the transporter encoded by the SLC25A48 gene. In the mitochondria choline is oxidized to betaine aldehyde by choline dehydrogenase (encoded by the CHDH gene). Betaine aldehyde is then oxidized to betaine by aldehyde dehydrogenase 7 family member A1 (encoded by the ALDH7A1 gene) which is also known as betaine aldehyde dehydrogenase. Following its synthesis, betaine is transported out of the mitochondria where is can function as a methyl donor in the synthesis of methionine from homocysteine.
The CHDH gene is located on chromosome 3p21.1 and is composed of exons 9 that encode a 594 amino acid protein. Befitting the role of betaine as an important osmolyte, the highest levels of expression of the CHDH gene are seen in the kidneys.
The ALDH7A1 gene is located on chromosome 5q23.2 and is composed of 18 exons that generate three alternatively spliced mRNAs, each of which encode a distinct protein isoform. Highest levels of expression of the ALDH7A1 gene are seen in the liver and kidneys.
The role of choline in betaine synthesis, and the subsequent principal role of betaine as a methyl donor, primarily in the context of the methionine cycle in the liver, makes this function of choline one of its most important. Betaine is also important as an osmolyte in the kidney.
In the methionine cycle the methyl group of betaine is transferred to homocysteine via the action of betaine-homocysteine S-methyltransferase encoded by the BHMT gene. Betaine-homocysteine S-methyltransferase is a cytosolic enzyme whose expression is restricted to the liver and kidneys.
The BHMT gene is located on chromosome 5q14.1 and is composed of 8 exons that encode a 406 amino acid protein. Humans express a second betaine-homocysteine S-methyltransferase enzyme (BHMT2) that is encoded by the BHMT2 gene located in the same region of chromosome 5 (5q14.1) as the BHMT gene.
Given that methionine synthase is ubiquitously expressed, the role of BHMT in homocysteine re-methylation outside of hepatocytes and kidneys is limited.
Cysteine Biosynthesis
In cysteine synthesis, homocysteine condenses with serine to produce cystathionine which is catalyzed by cystathionine β-synthase (cystathionine beta synthase: CBS). Cystathionine is subsequently cleaved by cystathionine γ-lyase (also called cystathionase) to produce cysteine and α-ketobutyrate. The sum of the latter two reactions is known as transsulfuration.
Both of these enzymes require pyridoxal phosphate (PLP: derived from vitamin B6) as a cofactor, and both are under regulatory control. Cystathionine γ-lyase is also negatively regulated by cysteine-mediated allosteric control. In addition, cysteine inhibits the expression of the cystathionine β-synthase gene.
Cysteine is used for protein synthesis and other body needs, while the α-ketobutyrate is first converted to propionyl-CoA and then via a 3-step process to the TCA cycle intermediate succinyl-CoA. While cysteine readily oxidizes to form the disulfide cystine, cells contain little if any free cystine because the ubiquitous reducing agent, glutathione, effectively reverses the formation of cystine by a non-enzymatic reduction reaction.
Cystathionine β-synthase is encoded by the CBS gene. The CBS gene is located on chromosome 21q22.3 and is composed of 23 exons which generate five alternatively spliced mRNAs that collectively encode two distinct proteins of 551 amino acids (isoform 1) and 446 amino acids (isoform 2).
Cystathionine γ-lyase is encoded by the CTH gene. The CTH gene is located on chromosome 1p31.1 and is composed of 13 exons that undergo alternative splicing generating three mRNAs, each of which encode distinct protein isoforms.
Genetic defects have been identified in both the CBS gene (see below) and the CTH gene. Missing or impaired cystathionine γ-lyase leads to excretion of cystathionine in the urine but does not have any other untoward effects. Rare cases are known in which cystathionine γ-lyase is defective and operates at a low level. This genetic disease leads to methioninuria with no other consequences.
Deficiency in B12, or folate, or defects in the MTR gene all will result in the development of megaloblastic anemias. The anemia develops due to the trapping of folate in the reduced form (N5-methyl-THF) which results in depletion of the active folate pool. The active folate pool is required for purine and thymine nucleotide biosynthesis needed for DNA synthesis. Bone marrow erythroblasts begin to enlarge in preparation for cell division but, due to loss of nucleotide synthesis, they remain as megaloblasts.
The Methionine Cycle
Given that methionine can be recycled in the context of conversion to, and utilization of, S-adenosylmethionine (SAM), the enzymes and intermediates in the methionine recycling constitute what is referred to as the methionine cycle. Although all of the enzymes and intermediates in this methionine cycle were discussed in the context of the methionine to cysteine conversion, the following Figure outlines the key features of the cycle.
Homocysteinemia / Homocystinuria
Homocysteinemias (commonly referred to as homocystinurias) represent a family of inherited disorders resulting from defects in several of the genes involved in the conversion of methionine to cysteine. As the name implies, these disorders result in elevated levels of homocysteine and homocystine in the blood, where the elevated urine output of the metabolite is referred to as homocysteinurina or homocystinuria. Homocystine is a disulfide-bonded homodimer of two homocysteines. This is similar to the formation of cystine from two cysteines as diagrammed in the Cystinuria page.
In addition to homocysteinuria/homocystinuria, patients excrete elevated levels of methionine and metabolites of homocysteine. The most common causes of homocystinuria (classic homocystinuria) are mutations in the gene (CBS) encoding cystathionine β-synthase. Homocystinuria is often associated with intellectual impairment, although the complete syndrome is multifaceted and many individuals with this disease are mentally normal, while others experience variable levels of developmental delay along with learning problems. Common symptoms of homocystinuria are dislocated optic lenses (ectopia lentis), osteoporosis, lengthening and thinning of the long bones, and an increased risk of abnormal blood clotting (thromboembolism). The nature of the lens dislocation (subluxation), evident in homocysteinemia patients, can serve as a differential diagnostic tool. Lens dislocation is also found in patients suffering from Marfan syndrome, which is a connective tissue disorder caused by mutations in the fibrillin gene (FBN1). In homocystinuria the lens subluxation occurs in a downward and inward direction, whereas in Marfan syndrome the lens subluxation occurs upward and outward. Some instances of genetic homocysteinemia respond favorably to pyridoxine therapy suggesting, that in these cases the defect in CBS is a decreased affinity for the cofactor, pyridoxal phosphate.
Homocysteinemia can also result from vitamin deficiencies due to the role of the co-factor forms of B6, B12, and folate in the overall metabolism of methionine. Vitamin B6 (as pyridoxal phosphate) is required for the activity of CBS and cystathionine γ-lyase and the homocysteinemia that results with B6 deficiency is also associated with elevated methionine levels in the blood. Vitamin B12 and folate (as N5-methyl-THF) are required for the methionine synthase reaction so a deficiency of either vitamin can result in homocysteinemia presenting with reduced levels of plasma methionine. The enzyme methylmalonyl-CoA mutase also requires B12 and so a homocysteinuria resulting from a deficiency in this vitamin is also associated with methylmalonic acidemia. Indeed, the measurement of serum methionine and methylmalonic acid in cases of homocysteinemia (homocystinuria) allows for a differential diagnosis of the nutritional (non-genetic) cause.
Another related disorder, referred to as hyperhomocysteinemia, is most often associated with manifestation of symptoms much later in life. The characteristic pathology of hyperhomocysteinemia is coronary artery disease (CAD) and an increased risk for deep vein thromboses, DVT. In addition, there is an increased risk for mild cognitive impairment and dementia. Evidence suggests a link between hyperhomocysteinemia and Alzheimer disease. Similar to the dietary causes of homocystinuria, hyperhomocysteinemia can result from deficiencies in vitamin B6, vitamin B12, and folate.
An inherited form of hyperhomocysteinemia results from mutations in the methylene tetrahydrofolate reductase (MTHFR) gene. The most common mutation in this gene that results in hyperhomocysteinemia is a C to T change at nucleotide position 677 (C677T). Mutations in the methionine synthase (MTR) gene are also associated with inherited hyperhomocysteinemia. In the case of the MTR gene the most common mutation is a change of an A for a G at nucleotide position 2756 (A2756G).
Elevated levels of homocysteine in the blood have been shown to correlate with cardiovascular dysfunction. The role of homocysteine in cardiovascular disease is related to its ability to induce a state of inflammation. Homocysteine serves as a negatively charged surface that attracts the contact phase of the intrinsic pathway of blood coagulation. Activation of the intrinsic coagulation cascade leads to inappropriate thrombolytic events as well as resulting in increases in inflammatory cytokine release from leukocytes that are activated as a result of the pro-coagulant state. Therefore, it is important to ensure that proper function of the methionine synthase reaction is maintained. Although it would be assumed that increased intake of vitamin B12 should lead to increased conversion of homocysteine to methionine and thus, reduced levels of circulating homocysteine, controlled studies have shown that this does not occur.
Functions of S-Adenosylmethionine (SAM or AdoMet)
S-adenosylmethionine (abbreviated SAM, SAMe, or AdoMet), synthesized as described above, is utilized in a wide array of methyl transfer reactions involving nucleic acids, lipids, proteins, neurotransmitters, and small molecules. During the course of the transfer of the methyl group from SAM to an appropriate acceptor, S-adenosylhomocysteine (abbreviated SAH or AdoHcy) is generated. The resultant SAH is catabolized to adenosine and homocysteine through the action of adenosylhomocysteinase (also called S-adenosylhomocysteine hydrolase) which is encoded by the AHCY gene.
The AHCY gene is located on chromosome 20q11.22 and is composed of 16 exons that generate six alternatively spliced mRNAs encoding three distinct proteins isoforms of 432 amino acids (isoform 1), 404 amino acids (isoform 2), and 434 amino acids (isoform 3). Mutations in the AHCY gene are one of the inherited causes of hypermethioninemia.
The role of SAM in nucleotide and protein methylation contributes to several epigenetic processes and points to the role of nutritional components, in this case methionine, in the control of gene expression. The DNA methyltransferases encoded by the DNMT1 and DNMT3 genes utilize SAM in the methylation of cytidine residues found in CpG dinucleotides in DNA. The RNA methyltransferase encoded by the RNMT gene utilizes SAM as a substrate for the N7-methylation of the guanine residue present in the mRNA 5′-cap structure. The RNA methyltransferases encoded by the METTL3 and METTL14 genes incorporate methyl groups into adenine and cytidine residues in mRNA molecules utilizing SAM as the methyl donor. These latter mRNA methylation events represent important posttranscriptional mechanisms for the regulation of gene expression.
Numerous SAM-dependent methyltransferases are involved in the methylation of histone proteins which represents another mode of epigenetic regulation of gene expression. These methyltransferases all utilize SAM as the methyl donor and incorporate the methyl group onto lysine residues, arginine residues, and histidine residues in proteins. Several histone lysine and histone arginine N-methyltransferases have been identified including all of the HMT (histone lysine methyltransferase) gene family enzymes and the PRMT (protein arginine methyltransferase) gene family enzymes.
All lysine methyltransferase enzymes belong to the large family of enzymes identified as the methyltransferase family. Humans express six large families of methyltransferases identified as the homocysteine methyltransferase family, the lysine methyltransferase family, the radical S-adenosylmethionine domain containing family, the seven-beta-strand methyltransferase motif containing family, the SET domain containing family, and the SPOUT methyltransferase domain containing family.
Many of the proteins that are targets for enzymes of the PRMT family are involved in the processes of signal transduction or regulation of transcription. In addition to members of the histone protein family and transcription factor family, numerous other proteins are subject to either lysine or arginine methylation.
Additional enzymes that utilize SAM as a methyl donor are involved in the modification of proteins that serve functions in diverse processes such as protein damage repair, protein stability, and protein function. The enzyme identified as protein-L-isoaspartate (D-aspartate) O-methyltransferase (encoded by the PCMT1 gene) is required for the repair of deamidated aspartate and asparagine residues in proteins and it utilizes SAM in these reactions.
The function of the enzyme, isoprenylcysteine carboxyl methyltransferase (encoded by the ICMT gene) is to methylate the cysteine residues in the C-terminus of proteins following their prenylation. Humans express three genes that catalyze the SAM-dependent cysteine methylation reaction on prenylated proteins with the ICMT gene being the most abundantly expressed. The SAM-dependent protein methyltransferase encoded by the LCMT1 gene (leucine carboxyl methyltransferase 1) catalyzes the methylation of a C-terminal leucine residue in the Ser/Thr phosphatase identified as PP2A, a modification required for its proper function. The synthesis of the diphthamide residue found on His715 in human translation elongation factor eEF2 requires a methylation step involving SAM as the methyl donor.
Numerous reaction pathways involved in the synthesis of small molecules, as well as the synthesis and catabolism of neurotransmitters involve enzymes that are SAM-dependent methyltransferases. These methyltransferases are classified as either N-methyltransferases or O-methyltransferases dependent on whether the acceptor of the methyl group is nitrogen or an oxygen, respectively. The synthesis of epinephrine from norepinephrine requires the SAM-dependent enzyme phenylethanolamine N-methyltransferase encoded by the PNMT gene. The conversion of serotonin to melatonin requires the enzyme acetylserotonin O-methyltransferase encoded by the ASMT gene. Creatine synthesis also requires SAM-dependent methylation in a reaction catalyzed by guanidinoacetate N-methyltransferase (encoded by the GAMT gene). The catabolism of the catecholamines, epinephrine, norepinephrine, and dopamine, involves the SAM-dependent enzyme catechol O-methyltransferase (encoded by the COMT gene). The catabolism of histamine occurs either through oxidation or methylation. The methylation pathway involves the SAM-dependent enzyme histamine N-methyltransferase which is encoded by the HNMT gene.
Lipid metabolism is another important process that involves SAM-dependent methylation. The conversion of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) requires the enzyme phosphatidylethanolamine N-methyltransferase (encoded by the PEMT gene) which carries out three successive SAM-dependent methylation reactions. This reaction is a critically important reaction of membrane lipid homeostasis. Lipid synthesis and remodeling is important in all cell membranes but is particularly critical in the homeostasis of the myelin sheath protecting neurons in the nervous system. Reduced capacity to carry out the methionine synthase reaction, due to nutritional or disease mediated deficiency of vitamin B12, results in reduced SAM production. In turn, reduced levels of SAM in the brain are a contributor to the neural degeneration (i.e. depression and peripheral neuropathy) seen in chronic B12 deficiency.
The metabolism and detoxification of numerous xenobiotic compounds is also known to require SAM-dependent methyltransferase family enzymes. The anti-cancer drugs of the thiopurine class, such as 6-mercaptopurine, are metabolized by the enzyme thiopurine S-methyltransferase which is encoded by the TPMT gene.
Tyrosine Biosynthesis
Tyrosine is produced in cells by hydroxylating the essential amino acid phenylalanine. This reaction is catalyzed by phenylalanine hydroxylase (PAH). This relationship is much like that between cysteine and methionine. Half of the daily requirement for phenylalanine is for the production of tyrosine; if the diet is rich in tyrosine itself, the requirements for phenylalanine can be reduced by about 50%.
Phenylalanine hydroxylase (PAH: more specifically phenylalanine 4-hydroxylase) is a mixed-function monooxygenase that is one of three enzymes belonging to the biopterin-dependent aromatic amino acid hydroxylase (AAAH) family. Phenylalanine hydroxylase is encoded by the PAH gene. The PAH gene is located on chromosome 12q23.2 and is composed of 15 exons that generate two alternatively spliced mRNAs, both of which encode the same 452 amino acid protein. The required biopterin is in the form of tetrahydrobiopterin (often designated BH4 or H4B).
Phenylalanine hydroxylase functions primarily as a homotetrameric enzyme but exists in cells in equilibrium with homodimeric forms as well. The PAH monomeric protein is composed of three functional domains. The N-terminal domain (amino acids 1-117) comprises the regulatory domain containing the Phe-binding subdomain. The catalytic domain comprises amino acids 118-427. The C-terminal domain (amino acids 428-452) is responsible for the oligomerization of the monomers.
The other two enzymes in the AAAH family are tyrosine hydroxylase and tryptophan hydroxylase. An additional clinically significant group of enzymes that requires tetrahydrobiopterin as cofactor are the nitric oxide synthase (NOS) family of enzymes.
Phenylalanine hydroxylase transfers one atom from molecular oxygen (O2) into the hydroxyl of tyrosine and the other hydroxylates tetrahydrobiopterin forming an intermediate identified as pterin 4α-carbinolamine (also called 4α-hydroxypterin or 4α-hydroxydihydrobiopterin).
Tetrahydrobiopterin can be regenerated in a salvage pathway that involves the enzymes pterin 4α-carbinolamine dehydratase 1 and dihydropteridine reductase.
The pterin 4α-carbinolamine dehydratase 1 enzyme is encoded by the PCBD1 gene. The PCBD1 gene is located on chromosome 10q22.1 and is composed of 6 exons that generate three alternatively spliced mRNA, each of which encode a distinct protein isoform. The PCBD1 gene does not contain the typical TATA-box and CAAT-box basal promoter elements found in the majority of mRNA encoding genes.
The product of the PCBD1 encoded enzyme, acting on pterin 4α-carbinolamine, is the quinoid molecule identified as dihydrobiopterin. In addition to the catalytic function of PCBD1, the protein serves as a transcriptional co-activator for two transcription factors of the hepatocyte nuclear factor-1 family, HNF-1α (encoded by the HNF1A gene) and HNF-1β (encoded by the HNF1B gene).
Dihydrobiopterin is then converted to tetrahydrobiopterin by the NADH-dependent enzyme commonly referred to as dihydropteridine reductase. Human dihydropteridine reductase is encoded by the QDPR (quinoid dihydropteridine reductase) gene. The QDPR gene is located on chromosome 4p15.32 and is composed of 7 exons that generate two alternatively spliced mRNAs encoding proteins of 244 amino acids (isoform 1) and 213 amino acids (isoform 2).
Missing or deficient phenylalanine hydroxylase results in hyperphenylalaninemia. Hyperphenylalaninemia is defined as a plasma phenylalanine concentration greater than 2 mg/dL (120 μM). The most widely recognized hyperphenylalaninemia (and most severe) is the genetic disease known as phenylketonuria (PKU). Patients suffering from PKU have plasma phenylalanine levels >1000 μM, whereas the non-PKU hyperphenylalaninemias exhibit levels of plasma phenylalanine <1000 μM.
Untreated PKU leads to severe intellectual impairment, however, the precise mechanism by which this enzyme deficiency leads to the severe neural degeneration is not fully understood. One theory suggests that the accumulation of phenylalanine interferes with the transport of tyrosine into the brain. A reduction in brain tyrosine levels would then result in reduced synthesis of the neurotransmitters dopamine and norepinephrine. Another theory suggests that the intellectual impairment is caused by the accumulation of phenylalanine in the brain, which becomes a major donor of amino groups in aminotransferase activity and depletes neural tissue of 2-oxoglutarate (α-ketoglutarate). This absence of 2-oxoglutarate in the brain shuts down the TCA cycle and the associated production of aerobic energy. Although both theories are plausible no direct evidence for either has been demonstrated.
The product of phenylalanine transamination, phenylpyruvic acid, is reduced to phenylacetate and phenyllactate, and all three compounds appear in the urine. The presence of phenylacetate in the urine imparts a “mousy” odor. If the problem is diagnosed early, the addition of tyrosine and restriction of phenylalanine from the diet can minimize the extent of intellectual impairment.
Because of the requirement for tetrahydrobiopterin in the function of phenylalanine hydroxylase, mutations in the PCBD1 gene or the QDPR gene can manifest with hyperphenylalaninemia. Mutations in the PCBD1 gene and the QDPR gene are causes of a family of disorders termed tetrahydrobiopterin deficiency syndromes. At least nine different mutations in the PCBD1 gene have been found to be associated with inherited tetrahydrobiopterin deficiency. However, the mutations in PCBD1 are not associated with significant pathology. It is thought that other enzymes can compensate for the reduced activity of PCBD1.
At least 30 different mutations in the QDPR gene are associated with inherited tetrahydrobiopterin deficiencies and these mutations account for approximately 30% of all the cases of these related disorders. Because the activity of the QDPR gene encoded enzyme is also involved in the functions of the tyrosine hydroxylase and tryptophan hydroxylase enzymes, mutations in the QDPR gene present as atypical hyperphenylalaninemias but are also associated with the potential for microcephaly, hypotonia, intellectual impairment, and convulsions due to neurotransmitter synthesis deficits. In these atypical hyperphenylalaninemias, that result from QDPR mutations, restriction of phenylalanine from the diet has no therapeutic benefits.
Tetrahydrobiopterin Synthesis
Tetrahydrobiopterin (6R-L-erythro-5,6,7,8-tetrahydrobiopterin) is most often abbreviated BH4 but also can be written as HB4. Depending upon cell type BH4 can be generated by several different (alternative) de novo biosynthesis pathways as well as by two different salvage pathways. The major de novo pathway is diagrammed in the Figure below. The synthesis of BH4 occurs de novo from the nucleotide, GTP. There are three principal enzymes involved in the major de novo pathway of BH4 synthesis. In reaction order these enzymes are GTP cyclohydrolase 1 (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS), and sepiapterin reductase (SPR).
GTP cyclohydrolase 1 is encoded by the GCH1 gene which is located on chromosome 14q22.2 and is composed of 9 exons that generate four alternatively spliced mRNAs that collectively encode three distinct protein isoforms.
The 6-pyruvoyltetrahydropterin synthase enzyme is encoded by the PTS gene which is located on chromosome 11q23.1 and is composed of 6 exons that encode a 145 amino acid protein.
Sepiapterin reductase is encoded by the SPR gene which is located on chromosome 2p13.2 and is composed of three exons that encode a 261 amino acid protein.
The reaction catalyzed by GTP cyclohydrolase 1 (GTPCH) is considered the rate-limiting and committed reaction of BH4 synthesis. Functional GTP cyclohydrolase 1 exists as a homodecamer that consists of two tightly associated dimers that each comprise a pentameric complex of the GTP cyclohydrolase 1 monomers. Through the actions of GTP cyclohydrolase 1 GTP is converted to 7,8-dihydroneopterin triphosphate.
The second step in BH4 synthesis, catalyzed by 6-pyruvoyltetrahydropterin synthase (PTPS), involves the Zn2+– and Mg2+-dependent rearrangement of 7,8-dihydroneopterin triphosphate, with concomitant loss of the triphosphate, to 6-pyruvoyltetrahydropterin. Functional 6-pyruvoyltetrahydropterin synthase is a homoheaxmeric complex that is formed via the head-to-head interaction of a pair of trimers.
The final step of BH4 synthesis actually involves three distinct steps all of which are catalyzed by the NADPH-dependent enzyme, sepiapterin reductase (SR). Functional sepiapterin reductase is a homodimeric enzyme which first converts 6-pyruvoyltetrahydropterin to 1′-hydroxy-2′-oxopropyltetrahydropterin, which is then reduced to 1′-oxo-2′-hydroxypropyltetrahydropterin, and then finally to 5,6,7,8-tetrahydrobiopterin, BH4.
Disorders Associated with Tetrahydrobiopterin Synthesis and Salvage
There are at least four identified disorders that manifest with BH4-deficient hyperphenylalaninemia designated as hyperphenylalaninemia, BH4-deficient type A (HPABH4A), HPABH4B, HPABH4C, and HPABH4D. Each of the four HPABH4 disorders are phenotypically characterized by hyperphenylalaninemia, depletion of the neurotransmitters dopamine and serotonin, and progressive cognitive and motor deficits.
Mutations in the PTS gene are the cause of the autosomal recessive disorder referred to as HPABH4A. HPABH4A represents the most common form of inherited tetrahydrobiopterin deficiency.
Mutations in the GCH1 gene are the cause of the autosomal recessive disorder referred to as HPABH4B.
Mutations in the QDPR gene, that encodes the enzyme commonly referred to as dihydrobiopterin reductase (DHPR), are the cause of the autosomal recessive disorder referred to as HPABH4C.
Mutations in the PCBD1 gene, that encodes the tetrahydrobiopterin salvage enzyme pterin 4α-carbinolamine dehydratase 1, are the cause of the autosomal recessive disorder referred to as HPABH4D.
Mutations in the SPR gene are associated with a form of DOPA-responsive dystonia. Inheritance of SPR deficiency may be autosomal recessive or autosomal dominant but has not been definitively characterized. Loss of the SPR encoded enzyme results in neurologic deterioration due to severe dopamine and serotonin deficiencies in the central nervous system.
Ornithine and Proline Biosynthesis
Glutamate is the precursor for the endogenous synthesis of both proline and ornithine with Δ1-pyrroline-5-carboxylate serving as a branch point intermediate leading to one or the other of these two amino acids. While ornithine is not one of the 20 amino acids used in protein synthesis, it plays a significant role as the acceptor of carbamoyl phosphate in the urea cycle. Indeed, the urea cycle represents another pathway that can replenish ornithine utilizing arginine. Ornithine serves an additional important role as the precursor for the synthesis of the polyamines.
The production of ornithine from glutamate is important when dietary arginine, the other principal source of ornithine, is limited. The synthesis of ornithine from glutamate occurs only in the intestines. The conversion of glutamate to Δ1-pyrroline-5-carboxylate is catalyzed by a bi-functional enzyme that is a member of the aldehyde dehydrogenase family. This enzyme, aldehyde dehydrogenase 18 family, member A1 (encoded by the ALDH18A1 gene), possess both γ-glutamyl kinase activity and an NADPH-dependent γ-glutamylphosphate reductase activity. The ALDH18A1 encoded enzyme is also known as delta-1-pyrroline-5-carboxylate synthase (P5CS).
The Δ1-pyrroline-5-carboxylate intermediate in this pathway can exist as the open-chain tautomeric compound, glutamate γ-semialdehyde, which can be transaminated to ornithine via the action of ornithine aminotransferase. Ornithine aminotransferase utilizes glutamate as the amino donor and releases 2-oxoglutarate (α-ketoglutarate).
The ALDH18A1 gene is located on chromosome 10q24.1 and is composed of 17 exons that generate ten alternatively spliced mRNAs that collectively encode six distinct protein isoforms. Mutations in the ALDH18A1 gene result is a disorder characterized by hyperammonemia, hypoornithinemia, hypocitrullinemia, hypoargininemia and hypoprolinemia. This disorder is also associated with CNS degeneration, connective tissue disruption, and cataract formation.
Ornithine aminotransferase is encoded by the OAT gene. The OAT gene is located on chromosome 10q26.13 and is composed of 13 exons that generate ten alternatively spliced mRNAs that collectively encode four distinct protein isoforms.
The fate of Δ1-pyrroline-5-carboxylate (and its open-chain tautomer, glutamate γ-semialdehyde) depends on prevailing cellular conditions. Ornithine production occurs from the semialdehyde compound via a simple glutamate-dependent transamination, producing ornithine. When arginine concentrations become elevated, the ornithine contributed from the urea cycle plus that from Δ1-pyrroline-5-carboxylate inhibit the aminotransferase reaction, with accumulation of Δ1-pyrroline-5-carboxylate as a result. The accumulating Δ1-pyrroline-5-carboxylate can then be reduced to proline by the NAD(P)H-dependent enzyme pyrroline-5-carboxylate reductase 1.
Pyrroline-5-carboxylate reductase 1 is encoded by the PYCR1 gene. The PYCR1 gene is located on chromosome 17q25.3 and is composed of 10 exons that generate six alternatively spliced mRNAs that collectively encode five different isoforms of the enzyme.
Serine Biosynthesis
The main pathway to de novo biosynthesis of serine starts with the glycolytic intermediate 3-phosphoglycerate. An NADH-linked dehydrogenase (phosphoglycerate dehydrogenase; also commonly called 3-phosphoglycerate dehydrogenase) converts 3-phosphoglycerate into a keto acid, 3-phosphohydroxypyruvate, suitable for subsequent transamination. The aminotransferase, phosphoserine aminotransferase 1, utilizing glutamate as a donor, produces 3-phosphoserine, which is converted to serine by phosphoserine phosphatase.
Phosphoglycerate dehydrogenase is encoded by the PHGDH gene which is located on chromosome 1p12 and is composed of 18 exons that generate a protein of 533 amino acids. Phosphoglycerate dehydrogenase is found in a complex with aldolase A and PFK-1 of the glycolytic pathway. The activity of this complex is regulated by the state of ubiquitylation. In the deubiquitylated state the complex enhances glucose metabolism and serine synthesis from glucose, a process that has been shown to be enhanced in cancer cells. The primary deubiquitylating (DUB) enzyme is encoded by the JOSD2 (josephin domain containing 2) gene.
Mutations in the PHGDH gene are associated with a rare autosomal recessive disorder characterized by presentation in infancy with microcephaly, intractable seizures, and little to no psychomotor development. Cranial MRI analysis shows hypomyelination and absence of white matter.
Phosphoserine aminotransferase 1 is encode by the PSAT1 gene located on chromosome 9q21.2 and is composed of 9 exons that generate two alternatively spliced mRNAs encoding isoform 1 (370 amino acids; also called PSAT1 beta: PSAT1β) and isoform 2 (324 amino acids; also called PSAT1 alpha: PSAT1α).
The phosphoserine phosphatase gene (symbol: PSPH) is located on chromosome 7p11.2 and is composed of 13 exons that encode a 225 amino acid protein. The location of the PSPH gene on chromosome 7p11.2 is in a region of chromosome 7 that is deleted in Williams-Beuren syndrome, WBS (also called Williams syndrome, WS). WBS is associated with multiple organ system involvement typically showing supravalvular aortic stenosis (SVAS), intellectual impairment, and distinctive facial features.
As indicated below, serine can be derived from glycine (and vice versa) by a single step reaction that involves serine hydroxymethyltransferase (SHMT; also called glycine hydroxymethyltransferase) and tetrahydrofolate (THF). Indeed, the interconversion of serine and glycine via the involvement of THF represents the major pathway for the generation of N5,N10-methylene-THF which of a member of the active pool of folate derivatives. N5,N10-methylene-THF is required for purine nucleotide and thymine nucleotide biosynthesis. Humans express two serine hydroxymethyltransferase genes, one is a cytosolic enzyme while the other is located in the mitochondria.
The cytosolic enzyme is derived from the SHMT1 gene located on chromosome 17p11.2 which is composed of 14 exons that generate three alternatively spliced mRNAs, each of which encode a distinct protein isoform.
The location of the SMHT1 gene (17p11.2) resides within the 3.7 Mb region of chromosome 17 that is deleted in Smith-Magenis syndrome, SMS, a disorder associated with behavioral problems, psychomotor impairment, growth delay, speech delay, brachycephaly, midface hypoplasia, and a hoarse voice.
The mitochondrial enzyme is derived from the SHMT2 gene located on chromosome 12q13.3 which is composed of 14 exons that generate five alternatively spliced mRNAs that collectively encode three distinct isoforms of the enzyme.
One of the major functions of the SHMT2 encoded enzyme is in mitochondrial thymidylate synthesis pathway via its role in glycine and tetrahydrofolate metabolism. Mitochondrial thymidylate synthesis is required to prevent uracil accumulation in mitochondrial DNA (mtDNA). Another important function of SHMT2 is in the generation of NADH via metabolism of serine.
Because serine can be generated from glycine it is correct to state that the same alternative precursors for glycine synthesis, choline, 4-hydroxpyruvate, and glyoxylate, are precursors for the synthesis of serine. However, these alternative pathways for glycine synthesis represent minor pathways. The main pathway for glycine synthesis, outlined in the next section, involves serine which is primarily derived from glucose as well as the nitrogen from glutamate.
Glycine Biosynthesis
The main pathway to glycine is a one-step reversible reaction catalyzed by serine hydroxymethyltransferase (SHMT). This enzyme is a member of the family of one-carbon transferases and is also known as glycine hydroxymethyltransferase. This reaction involves the transfer of the hydroxymethyl group from serine to the cofactor tetrahydrofolate (THF), producing glycine and N5,N10-methylene-THF.
As pointed out in the previous section, there are mitochondrial and cytosolic versions of serine hydroxymethyltransferase. The cytosolic enzyme is encoded by the SHMT1 gene on chromosome 17 and the mitochondrial enzyme is encoded by the SHMT2 gene on chromosome 12. The major glycine biosynthetic enzyme is the cytosolic form of SHMT.
Role of Choline, Glyoxalate, and 4-Hydroxyproline in Glycine Synthesis
Glycine can be derived from sources other than serine but these alternative pathways do not represent significant sources of the amino acid.
Choline, which can be acquired in the diet or derived from serine, is a minor source of glycine. The pathway of choline conversion to glycine takes place in the mitochondria and involves several distinct enzyme catalyzed reactions. Choline dehydrogenase, encoded by the CHDH gene, converts choline to betaine (trimethylglycine) aldehyde. Conversion of choline to betaine aldehyde may also be catalyzed by butyrylcholinesterase (encoded by the BCHE gene) which is also referred to as choline oxidase. Betaine aldehyde is then oxidized to betaine by aldehyde dehydrogenase 7 family member A1 (encoded by the ALDH7A1 gene). The ALDH7A1 encoded enzyme has also been referred to as betaine aldehyde dehydrogenase. Betaine is then converted to dimethylglycine in a transmethylation reaction involving a methyl acceptor, most significantly homocysteine, and the enzyme betaine-homocysteine S-methyltransferase (BHMT) as depicted in The Methionine Cycle section above. Dimethylglycine is oxidatively demethylated to sarcosine (N-methylglycine) by the FAD- and THF-dependent enzyme, dimethylglycine dehydrogenase (encoded by the DMGDH gene). Sarcosine is then converted to glycine in another FAD- and THF-dependent reaction catalyzed by sarcosine dehydrogenase (encoded by the SARDH gene).
Glyoxalate and 4-hydroxyproline (which is metabolized to glyoxalate and pyruvate) can also serve as precursors for glycine. The final reaction in this five step metabolic pathway involves the amino transferase encoded by the AGXT gene, alanine–glyoxylate and serine–pyruvate aminotransferase.
Although there have been reports that demonstrate that glycine can be derived from threonine in mammals via a pathway involving the enzyme L-threonine dehydrogenase, this pathway is of no functional significance in the adult human. The human gene (TDH) encoding L-threonine dehydrogenase is an expressed pseudogene that does not encode a functional enzyme. Due to mutations in the TDH gene there are only truncated proteins that would be non-functional since they have lost part of the NAD+ binding motif and the C-terminal domain that would be responsible for binding L-threonine.
Glycine as a Neurotransmitter
Glycine is involved in many anabolic reactions other than protein synthesis including the synthesis of purine nucleotides, heme, glutathione, creatine and serine. In addition, glycine functions in the central nervous system as an inhibitory neurotransmitter where it participates in regulating signals that process motor and sensory information that permit movement, vision and audition. Glycine is co-released with GABA which is the primary inhibitory neurotransmitter.
Glycine action as a neurotransmitter is a function of the amino acid binding to a specific receptor, GlyR. GlyR is a member of the nicotinicoid receptor superfamily that includes the GABAA receptor (GABAAR), the excitatory nicotinic acetylcholine receptors (nAChR) and the serotonin type 3 receptor (5HT3). GlyR is a heteromeric (pentameric) complex consisting of a complex of either three or four α-subunits and one β-subunit. There are four different α-subunit genes (GLRA1, GLRA2, GLRA3, GLRA4) and a single β-subunit gene (GLRB) in the human genome. The GlyR is a ligand-gated ionotropic receptor that is a chloride channel. In addition to glycine, the GlyR can be activated by several other small amino acids such as alanine and taurine.
Glycine is also involved in the modulation of excitatory neurotransmission exerted via glutamate binding to N-methyl-D-aspartate (NMDA) type glutamate receptors.
Serine and Glycine in Oxalate Metabolism
Generally, dicarboxylic acids generated via the ω-oxidation pathway, are degraded in the mitochondria as well as the peroxisomes by their respective fatty acid β-oxidation pathways. Of clinical significance is the short-chain dicarboxylic acid, oxalic acid (oxalate). Oxalate is a dicarboxylic acid composed of two carbon atoms and exits in solution as a dianion of the formula C2O42– (–OOC—COO–).
Oxalate is present in many foods such as spinach, chard, nuts, black pepper, poppy seeds, and also occurs as a result of the metabolism of ascorbic acid, and the amino acids glycine, serine, and 4-hydroxyproline as well as metabolism of ethanolamine. The majority (40%) of daily endogenous oxalate production comes from the metabolism of dehydroascorbate. Only around 0.1% of endogenous oxalate arises from glycine catabolism, whereas approximately 15% of the daily endogenous production of glyoxylate derives from 4-hydroxyproline. The origin of 4-hydroxyproline is either the diet or the degradation of collagen proteins.
Glyoxylate, the endogenous precursor to oxalate, is a byproduct of the metabolism of the amino acids 4-hydroxyproline, serine, and glycine. Conversion of 4-hydroxyproline to oxalate begins with its oxidation to Δ1-pyrroline-3-hydroxy-5-carboxylate by proline dehydrogenase 2 (encoded by the PRODH2 gene). Δ1-pyrroline-3-hydroxy-5-carboxylate is converted to 4-hydroxy-L-glutamate via the action of aldehyde dehydrogenase 4 family member A1 (encoded by the ALDH4A1 gene). The ALDH4A1 encoded enzyme has also been referred to as Δ1-pyrroline-3-hydroxy-5-carboxylate dehydrogenase (1P5CDH). 4-Hydroxy-L-glutamate is a substrate for deamination by aspartate transaminase (AST) which uses 2-oxoglutarate (α-ketoglutarate) as the amino acceptor, generating 4-hydroxy-2-oxoglutarate and glutamate. 4-Hydroxy-2-oxoglutarate is hydrolyzed into pyruvate and glyoxylate by 4-hydroxy-2-oxoglutarate aldolase 1 (encoded by the HOGA1 gene).
Hepatic glycine and pyruvate can be converted to glyoxylate and alanine via the action of the vitamin B6-dependent enzyme, alanine–glyoxylate and serine–pyruvate aminotransferase (encoded by the AGXT gene). The AGXT gene is expressed exclusively in hepatocytes and is localized to the peroxisomes. The AGXT enzyme can also convert glyoxylate and alanine to glycine and pyruvate.
The majority of serine catabolism involves its conversion to glycine, followed by glycine decarboxylase-mediated metabolism as detailed below. Alternatively, serine can be converted to 3-hydroxypyruvate via transamination of pyruvate catalyzed by alanine–glyoxylate and serine–pyruvate aminotransferase 2 (encoded by the AGXT2 gene). The 3-hydroxypyruvate can then be converted to glycoaldehyde via the action of glyoxylate and hydroxypyruvate reductase (encoded by the GRHPR gene). The glycoaldehyde is ultimately metabolized to glyoxylate.
Glyoxylate can be metabolized to oxalate by hydroxyacid oxidase 1 (encoded by the HAO1 gene; also called glycolate oxidase) as well as by lactate dehydrogenase. Excess glyoxylate will enhance the metabolism of glyoxylate to oxalate.
Oxalate is excreted in the urine and approximately 50% – 80% is derived from the diet. If levels of oxalate in the glomerular filtrate increase (hyperoxaluria) it can chelate Ca2+ ions forming the insoluble compound, calcium oxalate. Indeed, calcium oxalate crystals are the most common type of compound contributing to renal calculi (kidney stones; nephrolithiasis).
Hyperoxaluria
The hyperoxalurias are divided into two categories: the primary hyperoxalurias (PH) and the secondary hyperoxalurias. Primary hyperoxalurias are rare autosomal recessive disorders of glyoxylate metabolism that are associated with high oxalate production, primarily within the liver. There are three distinct forms of PH identified as PH1, PH2, and PH3 (or PH I, PH II, and PH III). The primary hyperoxalurias are associated with overproduction and excessive urinary excretion of oxalate. The excess urinary oxalate leads to recurrent urolithiasis and nephrocalcinosis. Glomerular filtration rates decline in PH due to progressive renal involvement leading to oxalate accumulation and systemic oxalosis that manifests with metabolic bone disease, anemia, and skin ulcerations.
Primary hyperoxaluria type 1 is the most common inherited form of hyperoxaluria, representing 70%-80% of all cases. PH 1 results from mutations in the peroxisomal specific enzyme alanine–glyoxylate and serine–pyruvate aminotransferase encoded by the AGXT gene. The AGXT gene is located on chromosome 2q37.3 and is composed of 11 exons that encode a 392 amino acid protein.
Primary hyperoxaluria type 2 accounts for around 10% of all cases. PH 2 results from mutations in glyoxylate and hydroxypyruvate reductase encoded by the GRHPR gene. The GRHPR gene is located on chromosome 9p13.2 and is composed of 12 exons that encode a 328 amino acid protein. Expression of the GRHPR gene is seen in most tissues with the highest levels in the liver and then the kidney.
Primary hyperoxaluria type 3 results from mutations in 4-hydroxy-2-oxoglutarate aldolase 1 encoded by the HOGA1 gene. The HOGA1 gene is located on chromosome 10q24.2 and is composed of 7 exons that generate two alternatively spliced mRNAs that encode proteins of 327 (isoform 1) and 164 (isoform 2) amino acids. The HOGA1 gene is predominantly expressed in the kidney and the enzyme is localized to the mitochondria.
Glutamine/Glutamate and Asparagine/Aspartate Catabolism
Glutaminase is an important kidney tubule enzyme involved in the process of renal ammoniagenesis. Glutaminase converts glutamine (predominantly derived from the liver but also from many other tissues) to glutamate and NH4+, with the NH4+ being excreted in the urine. Glutaminase activity is present in many other tissues in addition to the kidney, such as the liver, small intestine, and neurons where its role is nearly as significant as it is within the kidney tubule. The glutamate produced from glutamine is converted to 2-oxoglutarate (α-ketoglutarate) via the action of glutamate dehydrogenase (making glutamine a glucogenic amino acid) and yielding another mole of NH4+.
There are two distinct glutaminase genes in humans identified as GLS (encoding the GLS1 enzyme) and GLS2 (encoding the GLS2 enzyme). The GLS gene is located on chromosome 2q32.2 and is composed of 20 exons that generate two alternatively spliced mRNAs generating proteins of 669 amino acids (isoform 1) and 598 amino acids (isoform 2). These two GLS-derived isoforms are often referred to as glutaminase C (GAC) and kidney-type glutaminase (KGA) but are collectively the glutaminase 1 (GLS1) enzymes.
The GLS encoded isoforms of glutaminase are primarily expressed in the kidneys. GLS encoded kidney-type glutaminase is the 669 amino acid isoform 1 protein and GLS encoded glutaminase C is the 598 amino acid isoform 2 protein. The GLS2 gene encoded glutaminase was originally thought to be liver specific but is in fact expressed in numerous tissues and is important in the glutamate-glutamine cycle in the brain.
The GLS2 encoded glutaminase was originally characterized as dependent on inorganic phosphate (Pi) for activity and is, therefore, also referred to as phosphate-activated glutaminase, PAG. However, both the GLS gene encoded enzymes and the GLS2 encoded enzymes require phosphate for activity with GLS enzymes being more sensitive. The GLS2 gene is located on chromosome 12q13.3 and is composed of 19 exons that generate four alternatively spliced mRNAs that encode three different isoforms of the enzyme. The GLS encoded enzymes are inhibited by glutamate but the GLS2 encoded enzyme is not. The GLS2 encoded enzyme is activated by ammonia but the GLS encoded enzymes are not.
Asparaginase (see above) is also widely distributed within the body, where it converts asparagine into ammonia and aspartate. Aspartate can serve as an amino donor in transamination reactions yielding oxaloacetate, which follows the gluconeogenic pathway to glucose.
Glutamate and aspartate are important in collecting and eliminating amino nitrogen via glutamine synthetase and the urea cycle, respectively. The catabolic path of the carbon skeletons involves simple 1-step aminotransferase reactions that directly produce net quantities of a TCA cycle intermediate. The glutamate dehydrogenase reaction operating in the direction of 2-oxoglutarate (α-ketoglutarate) production provides a second avenue leading from glutamate to gluconeogenesis.
Alanine Catabolism
Alanine is also important in intertissue nitrogen transport as part of the glucose-alanine cycle (see above) that delivers waste nitrogen from skeletal muscle to the liver where it can be incorporated into urea. The alanine catabolic pathway involves a simple aminotransferase reaction that directly produces pyruvate. The transamination is carried out by alanine transaminase, ALT (also called alanine aminotransferase).
Generally, the pyruvate produced from alanine has two distinct fates that are controlled by the energy demands of the liver and the metabolic needs of the organism as a whole. When energy needs are high in hepatocytes the pyruvate is oxidized by the PDH complex (PDHc) to acetyl-CoA and diverted into the TCA cycle. Alternatively, during the fasted state when blood glucose levels are low, the pyruvate is diverted into the gluconeogenic pathway so that the liver can release glucose to the blood. This makes alanine a glucogenic amino acid.
Arginine, Ornithine and Proline Catabolism
The catabolism of arginine begins within the context of the urea cycle. It is ultimately hydrolyzed to urea and ornithine by arginase.
Ornithine, in excess of urea cycle needs, is transaminated to form glutamate γ-semialdehyde which is in equilibrium with its tautomeric compound, Δ1-pyrroline-5-carboxylate. Catabolism of Δ1-pyrroline-5-carboxylate back to glutamate is catalyzed by aldehyde dehydrogenase 4 family, member A1 (also called delta-1-pyrroline-5-carboxylate dehydrogenase, P5CDH).
Aldehyde dehydrogenase 4 family, member A1 is encoded by the ALDH4A1 gene. The ALDH4A1 gene is located on chromosome 1p36.13 and is composed of 16 exons that generate four alternatively spliced mRNAs, two of which encode the same protein.
Proline catabolism involves a two-step process that is essentially a reversal of its synthesis process outlined above. Proline is first converted back to Δ1-pyrroline-5-carboxylate by the enzyme proline dehydrogenase 1.
Proline dehydrogenase 1 is encoded by the PRODH gene. The PRODH gene is located on chromosome 22q11.21 and is composed of 15 exons that generate two alternatively spliced mRNAs encoding precursor proteins of 600 amino acids (isoform 1) and 492 amino acids (isoform 2). As for the catabolism of ornithine, the resulting Δ1-pyrroline-5-carboxylate is converted to glutamate via the action of ALDH4A1.
The glutamate that results from ornithine and proline catabolism can then be converted to 2-oxoglutarate (α-ketoglutarate) in a transamination reaction. Therefore, ornithine and proline are both glucogenic. Since arginine is metabolized to urea and ornithine, and the resulting ornithine is a glucogenic precursor, arginine is also a glucogenic amino acid.
Serine Catabolism
The catabolism of serine in humans occurs in both the cytosol and the mitochondria. In both cellular compartments serine catabolism involves the conversion to glycine and then glycine oxidation to CO2 and NH3, with the production of two equivalents of N5,N10-methyleneTHF, as was described above in the section on glycine biosynthesis.
Serine can also be catabolized back to the glycolytic intermediate, 3-phosphoglycerate, by a pathway that is essentially a reversal of serine biosynthesis, however, the enzymes are different.
Serine (and glycine) can also be oxidized to oxalate (see also the Lipolysis and Fatty Acid Oxidation page) through a series of reactions proceeding to 3-hydroxypyruvate which involves transamination of pyruvate. The 3-hydroxypyruvate can then converted to glycoaldehyde via the action of glyoxylate and hydroxypyruvate reductase (encoded by the GRHPR gene). The glycoaldehyde is ultimately metabolized to glyoxylate. Glyoxylate can be metabolized to oxalate by hydroxyacid oxidase 1 (encoded by the HAO1 gene; also called glycolate oxidase). Alternatively, glyoxylate and alanine can be converted to glycine and pyruvate, respectively via the action of the vitamin B6-dependent enzyme, alanine–glyoxylate and serine–pyruvate aminotransferase (encoded by the AGXT gene).
Although it has been demonstrated in mammals, such as rodents and dogs, that serine can be converted to pyruvate through a deamination reaction catalyzed by the vitamin B6-dependent enzyme, serine/threonine dehydratase (encoded by the SDS gene), the serine deamination activity of the enzyme appears to be lacking in humans even though the activity is involved in the catabolism of threonine in humans as described below.
Role of Serine Catabolism in NADH Production
Serine catabolism in the mitochondria provides a mechanism for the generation of NADH independent of the TCA cycle. Serine is first converted to glycine via the mitochondrial serine hydroxymethyltransferase 2 (SHMT2) catalyzed reaction. The methylene-THF that is generated is oxidized to 10-formyl-THF via the methylene tetrahydrofolate dehydrogenase 2 (MTHFD2) catalyzed reaction which simultaneously reduces NAD+ to NADH. The MTHFD2 enzyme is resistant to inhibition by NADH and so this reaction can contribute to significant mitochondrial NADH production under increased serine catabolism as well as during periods where the major NADH producing catabolic pathways are inhibited by increasing NADH levels.
Accumulation of NADH is toxic to mitochondria via its contribution to the production of reactive oxygen species (ROS), superoxide anion and hydrogen peroxide. Serine catabolism can, therefore, contribute to mitochondrial ROS production which can precipitate mitochondrial apoptosis. During hypoxic conditions serine catabolism contributes to NADH production and this may contribute to tumor progression. However, there are limits to this benefit as enhanced serine metabolism-mediated NADH production contributes to ROS-mediated toxicity in these cells. Indeed, this pathway is currently being studies as a means to pharmacologically enhance serine catabolism in cancer cells to mediate their programmed death. Conversely, pharmacologic inhibition of mitochondrial serine catabolism could prove to be beneficial in chronic ischemia conditions such as cardiovascular disease.
Glycine Catabolism
Glycine is classified as a glucogenic amino acid, since it can be converted to serine by serine hydroxymethyltransferase, and serine can be converted back to the glycolytic intermediate, 3-phosphoglycerate or to pyruvate by serine/threonine dehydratase, although the latter reaction (serine to pyruvate) appears not be occur in human tissues. Nevertheless, the main glycine catabolic pathway leads to the production of CO2, ammonia, and one equivalent of N5,N10-methyleneTHF by the mitochondrial enzyme, glycine dehydrogenase (decarboxylating) which is also called the glycine cleavage complex, GCC.
The GCC is composed of four mitochondrial proteins encoded by four genes. The protein components of the GCC are the actual glycine decarboxylase subunit (identified as the P subunit: pyridoxal phosphate-dependent), a lipoic acid-containing subunit (the H subunit), a tetrahydrofolate-requiring enzyme called aminomethyltransferase (the T subunit), and dihydrolipoamide dehydrogenase (the L subunit).
The P subunit is encoded by the GLDC gene. The GLDC gene is located on chromosome 9p24.1 and is composed of 25 exons that encode a precursor protein of 1020 amino acids.
The H subunit is encoded by the GCSH gene. The GCSH gene is located on chromosome 16q23.2 and is composed of 6 exons that generate encode a protein of 173 amino acids.
The T subunit (aminomethyltransferase) is encoded by the AMT gene. The AMT gene is located on chromosome 3p21.31 and is composed of 9 exons that generate four alternatively spliced mRNAs generating four isoforms of the enzyme.
The L subunit is encoded by the DLD gene. The DLD gene is located on chromosome 7q31.1 and contains 14 exons that generate four alternatively spliced mRNAs, each of which encode a distinct protein isoform. The DLD encoded protein is also found as a subunit of several other important dehydrogenase complexes: the pyruvate dehydrogenase complex (PDHc), the 2-oxoglutarate (α-ketoglutarate) dehydrogenase complex, and the branched-chain keto-acid dehydrogenase (BCKD) complex.
Glycine Encephalopathy
Deficiencies in the H, P, or T proteins of glycine decarboxylase result in glycine encephalopathy (GCE) which is characterized by nonketotic hyperglycinemia (NKH). Several forms of GCE have been characterized with the neonatal phenotype being the most common.
In the neonatal form of GCE symptoms appear in the first few days of life and include lethargy, hypotonia, myoclonic jerks, and often death. Neonates that regain spontaneous respiration will develop intractable seizures and profound intellectual impairment.
In the infantile form of GCE, patients are seemingly normal for the first 4-6 months of life and then develop seizures and varying degrees of intellectual impairment.
In the mild-episodic form of GCE, patients present in childhood with mild intellectual impairment. These children will also develop episodes of delirium, chorea, and vertical gaze palsy during a febrile illness.
In the late-onset form of GCE, patients present in childhood with progressive spastic diplegia and optic atrophy, but intellectual function is preserved.
Threonine Catabolism
Threonine Catabolism via Serine Dehydratase
There are at least three pathways for threonine catabolism that have been identified in yeasts, insects, and vertebrates including mammals. The principal threonine catabolizing pathway in humans involves a glycine-independent serine/threonine dehydratase (also known as serine dehydratase/threonine deaminase) yielding ammonium ion (NH4+) and α-ketobutyrate (2-ketobutyrate). The 2-ketobutyrate is converted to propionyl-CoA via the addition of Co-A catalyzed via branched-chain keto acid dehydrogenase (BCKD).
Serine/threonine dehydratase is derived from the SDS gene. The SDS gene is located on chromosome 12q24.13 and is composed of 8 exons that encode a protein of 328 amino acids. Serine/threonine dehydratase is expressed at high levels only in the liver. It appears that in newborn infants catabolism of threonine occurs exclusively via the action of the serine/threonine dehydratase. Therefore, it is presumed that this is the predominant threonine catabolizing pathway in humans.
The resulting propionyl-CoA is converted, via a series of three reactions, to succinyl-CoA. These reactions take place within the mitochondria. The enzymes required for this conversion are propionyl-CoA carboxylase, methylmalonyl-CoA epimerase, and methylmalonyl-CoA mutase, respectively. Propionyl-CoA carboxylase is called an ABC enzyme due to the requirements for ATP, Biotin, and CO2 for the reaction. This propionyl-CoA conversion pathway is also required for the metabolism of the amino acids valine, isoleucine, and methionine, and odd-chain length fatty acids. For this reason this three-step reaction pathway is often remembered by the mnemonic as the VOMIT pathway, where V stands for valine, O for odd-chain fatty acids, M for methionine, I for isoleucine, and T for threonine.
Propionyl-CoA carboxylase functions as a heterododecameric enzyme (subunit composition: α6β6) and the two different subunits are encoded by the PCCA and PCCB genes, respectively. The PCCA gene is located on chromosome 13q32.3 and is composed of 32 exons that generate eleven alternatively spliced mRNAs, each of which encode a unique protein isoform. The PCCB gene is located on chromosome 3q22.3 and is composed of 17 exons that generate two alternatively spliced mRNAs encoding proteins of 539 amino acids (isoform 1) and 559 amino acids (isoform 2). Mutations in either the PCCA gene or the PCCB gene result in the organic acidemia known as propionic acidemia.
Methylmalonyl-CoA epimerase is encoded by the MCEE gene located on chromosome 2p13.3 and is composed of 4 exons that encode a 176 amino acid precursor protein. Methylmalonyl-CoA mutase is encoded by the MMUT gene located on chromosome 6p12.3 and is composed of 13 exons that encode a precursor protein of 750 amino acids. Mutations in the MMUT gene are one cause of the methylmalonic acidemias.
Threonine Catabolism via Serine Hydroxymethyltransferase (SHMT)
The second pathway of threonine catabolism utilizes serine hydroxymethyltransferase (SHMT). As indicated above in the Glycine Biosynthesis section, humans express two serine hydroxymethyltransferase genes, SHMT1 and SHMT2. The encoded enzymes belong to the family of one-carbon transferases. These enzymes are also identified as glycine hydroxymethyltransferase (was also identified as threonine aldolase). The products of SHMT action on threonine are acetaldehyde and glycine. The glycine can be converted to serine via the same enzyme and the serine is then catabolized as described above yielding pyruvate and NH4+. Thus, via this catabolic pathway threonine yields ketogenic and glucogenic byproducts.
In humans it appears that threonine aldolase is actually encoded by a non-functional pseudogene, whereas in other mammals and vertebrates (e.g. mice, zebrafish, and clawed frogs) the threonine aldolase gene encodes a functional threonine catabolizing enzyme.
Threonine Catabolism via Threonine Dehydrogenase (non-functional in humans)
An additional pathway of threonine catabolism in mammals occurs in the mitochondria and is initiated by threonine dehydrogenase (TDH) yielding α-amino-β-ketobutyrate (2-amino-3-ketobutyrate). The 2-amino-3-ketobutyrate is either converted to acetyl-CoA and glycine, via the action of 2-amino-3-ketobutyrate coenzyme A ligase (also called glycine C-acetyltransfease), or it can spontaneously degrade to aminoacetone which is converted to pyruvate.
The threonine dehydrogenase gene in humans appears to be an expressed non-functional pseudogene due to the incorporation of three inactivating mutations. Due to these mutations there are only truncated proteins produced that would be non-functional since they have lost part of the NAD+ binding motif and the C-terminal domain that would be responsible for binding L-threonine. Thus, whereas, this enzyme is a major threonine catabolizing enzyme in other mammals such as mice, it is the serine/threonine dehydratase enzyme that is most important for threonine catabolism in humans.
Cysteine Catabolism
There are several pathways for non-protein disposition of cysteine that include both metabolism and catabolism. The major cysteine catabolic pathway in humans occurs via the action of cysteine dioxygenase type 1 which oxidizes the sulfhydryl group of cysteine to sulfinate, producing the intermediate cysteine sulfinate.
Cysteine dioxygenase type 1 is encoded by the CDO1 gene. The CDO1 gene is located on chromosome 5q22.3 and is composed of 8 exons that generate four alternatively spliced mRNAs, each of which encode a distinct protein isoform.
Catabolism of cysteine sulfinate proceeds through transamination (catalyzed by cytosolic AST) to β-sulfinylpyruvate (3-sulfinylpyruvate) which then undergoes desulfuration yielding pyruvate and bisulfite, (HSO3–) which is in equilibrium with sulfite (SO32–) at pH 7.2. The enzyme, sulfite oxidase, then catalyzes the conversion of sulfite to sulfate. Sulfite oxidase is somewhat unique in that it uses the oxygen atom from H2O to convert sulfite to sulfate, (SO42–) and releases two protons (H+).
Sulfite oxidase is encoded by the SUOX gene. The SUOX gene is located on chromosome 12q13.2 and is composed of 8 exons that generate three alternatively spliced mRNAs, each of which encode the same 545 amino acid protein.
The enzyme cysteine desulfurase is another important enzyme associated with cysteine catabolism. Cysteine desulfurase removes the sulfur from cysteine yielding alanine. The sulfur remains associated with cysteine desulfurase and is subsequently transferred to numerous enzymes that possess iron-sulfur clusters for their activity. Cysteine desulfurase is a member of the pyridoxal phosphate (B6)-dependent aminotransferase family.
Cysteine desulfurase is encoded by the NFS1 gene. The NFS1 gene is located on chromosome 20q11.22 and is composed of 14 exons. In addition to alternative splicing (generating two mRNAs), there are alternative in-frame translational start sites in the NFS1 derived mRNAs. The use of these alternative translational start sites generates mitochondrial, cytoplasmic, and nuclear forms of the enzyme.
Other than protein, the most important metabolic products derived from cysteine are glutathione (GSH), the bile salt modifying compound, taurine, and as a source of the sulfur for coenzyme-A synthesis.
The enzyme cystathionase (cystathionine γ-lyase) can also transfer the sulfur from one cysteine to another generating thiocysteine and pyruvate. Transamination of cysteine yields β-mercaptopyruvate which then reacts with sulfite, (SO32–), to produce thiosulfate, (S2O32–) and pyruvate. Both thiocysteine and thiosulfate can be used by sulfurtransferases to incorporate sulfur into cyanide ion, (CN–), thereby detoxifying the cyanide to thiocyanate. These sulfurtransferase enzymes contain domains called rhodanese domains since they were first identified in a mitochondrial enzyme that was originally called rhodanese (thiosulfate sulfurtransferase, TST). Another rhodanese domain-containing enzyme, that is located in the cytosol, is called mercaptopyruvate sulfurtransferase (MPST). MPST transfers the sulfur group from 3-mercaptopyruvate to CN– and other thiol compounds.
Taurine Biosynthesis and Functions
Taurine can be acquired in the diet or synthesized from cysteine as outline above. Taurine is utilized in the synthesis of the bile acid conjugates taurocholate and taurochenodeoxycholate. Taurine also serves numerous other important functions in the body. Taurine is one of the most abundant amino acids in skeletal muscle, brain, and retina. In the retina taurine functions in photoreceptor development. In the brain taurine serves as a cytoprotectant against stress-related neuronal damage and other pathological conditions. Taurine is also an organic osmolyte involved in the regulation of cell volume. Taurine also plays important roles in the modulation of intracellular free calcium concentration and in the protection from mitochondrial stress.
Taurine synthesis occurs primarily in the liver since this is the only bile acid synthesizing tissue in the body. The other major tissues carrying out de novo taurine biosynthesis are the brain (astrocytes and neurons) and the retina. Taurine is derived from the cysteine catabolism intermediate, cysteine sulfinate. Cysteine sulfinate is converted to hypotaurine by the rate-limiting enzyme in taurine synthesis, cysteine sulfinic acid decarboxylase (also called sulfinoalanine decarboxylase).
Cysteine sulfinic acid decarboxylase is encoded by the CSAD gene. The CSAD gene is located on chromosome 12q13.3 and is composed of 23 exons that generate three alternatively spliced mRNAs each of which encode a unique isoform of the enzyme. Oxidation of hypotaurine to taurine is thought to occur spontaneously (non-enzymatically) although it may involve an as yet unidentified hypotaurine dehydrogenase. Evidence suggests that the catalytic conversion of hypotaurine to taurine is the function of enzyme encoded by the FMO1 (flavin containing dimethylaniline monoxygenase 1) gene.
Following its synthesis in the liver, taurine is transported to the blood where it is disseminated to other tissues of the body. Although neurons in the brain and cells of the retina can synthesize taurine, the majority of taurine in these tissues is derived from hepatic synthesis. Uptake of taurine into cells is mediated by the SLC family transporter, referred to as TauT, which is encoded by the SLC6A6 gene. Within the brain the SLC6A6 encoded transporter is primarily expressed in astrocytes but is also found at lower levels in neurons. Uptake of taurine into specific cells in the brain also occurs through the action of the GABA transporter encoded by the SLC6A13 gene.
Taurine exists in its zwitterionic form within the range of physiological pH. This biochemical characteristic of taurine allows for it to serve in the regulation of osmolarity. Indeed, taurine synthesis has been shown to be stimulated in cultured neurons exposed to hypertonic conditions. In addition, hyperosmolarity is associated increased expression of the SLC6A6 gene allowing for increased taurine uptake. Conversely, when cultured cells are exposed to hypo-osmotic conditions they transport taurine out to the surrounding medium.
Taurine plays a role in neurotransmitter by serving as a modulator of inhibitory neurotransmission. Taurine has been shown to interact with GABA receptors and glycine receptors. Indeed, evidence indicates that taurine is a potent ligand of the glycine receptor. Taurine is involved in the regulation of both cytoplasmic and intra-mitochondrial Ca2+ homeostasis. These effects of taurine allow it to function in the regulation of glutamate-mediated neurotransmission by reducing glutamate-induced Ca2+ transients in neurons.
Taurine is involved in mitochondrial homeostasis via its ability to act as a mitochondrial matrix pH buffer, by regulating mitochondrial potential, by modulating Ca2+-induced mitochondrial swelling, and by regulating the activity of mitochondrial dehydrogenases and ATP concentration. By exerting all of these effects, taurine helps to preserve normal mitochondrial physiology. These function of taurine are extremely important to overall brain function given that mitochondrial activity in neurons and astrocytes is crucial for brain activity.
Taurine function in overall mitochondrial homeostasis is also exerted at the level of apoptosis. With respect to brain function, the ability of taurine to reduce apoptosis in response to numerous noxious stimuli leads to improved brain outcomes following ischemic trauma. With respect to stress induced pathologies, taurine has also been shown to prevent endoplasmic reticulum (ER) stress. Taurine has been shown to attenuate mitochondrial apoptosis through inhibition of reductions in the level of the anti-apoptotic protein, Bcl-xL, and to prevent increases in the pro-apoptotic protein, Bax. In addition, taurine, prevents cytochrome c release from the mitochondria and inhibits the activation of the executioner caspase, caspase-3.
Synthesis of 3′-Phosphoadenosine-5′-Phosphsulfate: PAPS
One of the important uses of the sulfate that is derived from the catabolism of cysteine is as a precursor for the formation of 3′-phosphoadenosine-5′-phosphosulfate, (PAPS). PAPS is used for the transfer of sulfate to biological molecules such as the sugars of the glycosphingolipids.
Methionine Catabolism
The principal fates of the essential amino acid methionine are incorporation into polypeptide chains, and use in the production of cysteine and α-ketobutyrate via the reaction pathway involving the synthesis of SAM and cysteine as described above. The transulfuration reactions that produce cysteine from homocysteine and serine also produce α-ketobutyrate, the latter being converted first to propionyl-CoA.
In the catabolism of methionine the α-ketobutyrate is converted to propionyl-CoA. The propionyl-CoA is converted, via a mitochondrially-localized three reaction ATP-dependent pathway, to succinyl-CoA. The succinyl-CoA can then enter the TCA cycle for further oxidation. The enzymes required for this conversion are propionyl-CoA carboxylase, methylmalonyl-CoA epimerase, and methylmalonyl-CoA mutase, respectively. Propionyl-CoA carboxylase is called an ABC enzyme due to the requirements for ATP, Biotin, and CO2 for the reaction. The clinical significance of methylmalonyl-CoA mutase in this pathway is that it is one of only two enzymes that requires a vitamin B12-derived co-factor for activity. The other B12-requiring enzyme is methionine synthase (see the Cysteine, Methionine, and S-Adenosylmethionine Biosynthesis section above). This propionyl-CoA conversion pathway is also required for the metabolism of the amino acids valine, isoleucine, and threonine and fatty acids with an odd number of carbon atoms. For this reason this three-step reaction pathway is often remembered by the mnemonic as the VOMIT pathway, where V stands for valine, O for odd-chain fatty acids, M for methionine, I for isoleucine, and T for threonine.
Propionyl-CoA carboxylase functions as a heterododecameric enzyme (subunit composition: α6β6) and the two different subunits are encoded by the PCCA and PCCB genes, respectively. The PCCA gene is located on chromosome 13q32.3 and is composed of 32 exons that generate eleven alternatively spliced mRNAs, each of which encode a unique protein isoform. The PCCB gene is located on chromosome 3q22.3 and is composed of 17 exons that generate two alternatively spliced mRNAs encoding proteins of 539 amino acids (isoform 1) and 559 amino acids (isoform 2). Mutations in either the PCCA gene or the PCCB gene result in the organic acidemia known as propionic acidemia.
Methylmalonyl-CoA epimerase is encoded by the MCEE gene located on chromosome 2p13.3 and is composed of 4 exons that encode a 176 amino acid precursor protein. Methylmalonyl-CoA mutase is encoded by the MMUT gene located on chromosome 6p12.3 and is composed of 13 exons that encode a precursor protein of 750 amino acids. Mutations in the MMUT gene are one cause of the methylmalonic acidemias.
Regulation of the methionine metabolic pathway is based on the availability of methionine and cysteine. If both amino acids are present in adequate quantities, SAM accumulates and is a positive effector on cystathionine β-synthase, encouraging the production of cysteine and α-ketobutyrate (both of which are glucogenic). However, if methionine is scarce, SAM will form only in small quantities, thus limiting cystathionine β-synthase activity. Under these conditions accumulated homocysteine is remethylated to methionine, using N5-methyl-THF as the methyl donor.
Leucine, Isoleucine, and Valine Catabolism: Branched-Chain Amino Acids (BCAA)
This group of essential amino acids is identified as the branched-chain amino acids, BCAA. Because this arrangement of carbon atoms cannot be made by humans, these amino acids are essential in the diet. Indeed, the three BCAA represent 35%-40% of the total of the nine essential amino acids in the human diet and account for approximately 15% of the total of the amino acid composition of skeletal muscle.
The catabolism of all three amino acids occurs in most cells but the highest rates of catabolism takes place in skeletal muscle. BCAA catabolism yields both NADH and FADH2 which can be utilized for ATP generation which is a primary reason for their high rates of catabolism in skeletal muscle. The catabolism of all three of these amino acids uses the same enzymes in the first two steps. The first step in each case is a transamination using a pyridoxal phosphate-dependent BCAA aminotransferase (termed a branched-chain aminotransferase, BCAT), with 2-oxoglutarate (α-ketoglutarate) as amine acceptor.
Branched-Chain Aminotransferase: BCAT
Humans express two genes that encode BCAT activity. These two genes are identified as BCAT1 and BCAT2. The primary protein encoded by the BCAT1 gene is a cytosolic version of the enzyme and the protein is identified as BCATc. The primary protein encoded by the BCAT2 gene is a mitochondrial version of the enzyme and this protein is designated BCATm.
The BCAT1 gene is located on chromosome 12p12.1 and is composed of 14 exons that generate five alternatively spliced mRNAs, each of which encode a distinct protein isoform. BCAT1 isoform 1 is a 386 amino acid protein. BCAT1 isoform 2 is a 349 amino acid protein. BCAT1 isoform 3 is a 325 amino acid protein. BCAT1 isoform 4 is a 398 amino acid protein. BCAT1 isoform 5 is a 385 amino acid protein. Expression of the BCAT1 gene is restricted to only a few tissues types. The BCAT1 gene represents the primary BCAT expressing gene in the brain.
The BCAT2 gene is located on chromosome 19q13.33 and is composed of 13 exons that generate three alternatively spliced mRNAs that encode three different isoforms. BCAT2 isoform a is a 392 amino acid protein which is also referred to as PP18a. BCAT2 isoform b is a 300 amino acid protein which is also referred to as PP18b. The isoform b protein is found in the cytosol. BCAT2 isoform c is a 352 amino acid protein. Expression of BCAT2 is widely distributed among numerous tissues.
Although detectable in the fetal liver, the adult liver does not express either BCAT gene. Because BCAT is not present in hepatocytes, dietary BCAA, as well as gut microbiota-derived BCAA, pass through the liver and are deaminated by BCAT in other tissues, predominantly within skeletal muscle. The branched-chain keto acids (BCKA), that are the products of BCAT activity, if not fully oxidized in the tissues in which they are generated, will be delivered, via the blood, to the liver where they are oxidized.
The metabolism of the branched-chain amino acids is critical to overall nitrogen homeostasis in the brain and to the maintenance of proper levels of the excitatory neurotransmitter, glutamate. The critical enzymes that are necessary for this homeostatic process are the BCATc and BCATm isoforms of the BCAA aminotransferases. Within the brain different populations of cells express predominantly BCATc while others express predominantly BCATm and this differential distribution is what is important in overall neuronal nitrogen homeostasis.
As a result of the BCAT reaction, three different α-keto acids are produced and are oxidized using a common branched-chain α-keto acid dehydrogenase (BCKD) complex, yielding the three different CoA derivatives. Subsequently the metabolic pathways diverge, producing many intermediates. The BCKD complex is one of three dehydrogenase complexes whose vitamin-derived cofactor requirements can be remembered by the mnemonic: Tender Loving Care For Nancy, where the T stands for thiamine, the L for lipoic acid, the C for coenzyme A (CoA), the F for riboflavin, and the N for niacin. The other two dehydrogenase complexes are the PDHc and the 2-oxoglutarate dehydrogenase complexes associated with the TCA cycle.
Branched-Chain Keto Acid Dehydrogenase: BCKD
The BCKD complex is a multimeric enzyme composed of three catalytic subunits. The E1 portion of the complex is a thiamine pyrophosphate (TPP)-dependent decarboxylase with a heterotetrameric subunit structure of α2β2. The E2 portion is a dihydrolipoamide branched-chain transacylase composed of 24 lipoic acid-containing polypeptides. The E3 portion is a homodimeric flavoprotein identified as dihydrolipoamide dehydrogenase, DLD.
The E1α gene (symbol: BCKDHA) is located on chromosome 19q13.2 and contains 9 exons that generate two alternatively spliced mRNAs that encode alpha subunit isoform 1 (445 amino acids) and alpha subunit isoform 2 (444 amino acids).
The E1β gene (symbol: BCKDHB) is located on chromosome 6q14.1 and is composed of 21 exons that generate three alternatively spliced mRNAs that collectively encode a 392 amino acid protein (isoform 1 ) and a 322 amino acid protein (isoform 2).
The E2 gene (symbol: DBT) is located on chromosome 1p21.2 and contains 14 exons that that generate three alternatively spliced mRNAs that collectively encode a 482 amino acid precursor protein (isoform 1 ) and a 3301 amino acid protein (isoform 2).
The E3 gene (symbol: DLD) is located on chromosome 7q31.1 contains 14 exons that generate four alternatively spliced mRNAs, each of which encode a distinct protein isoform. The DLD gene encodes the same dihydrolipoamide dehydrogenase subunits found in the PDHc and the 2-oxoglutarate dehydrogenase (α-ketoglutarate dehydrogenase) complexes.
Regulation of BCKD Activity
Regulation of BCKD activity is exerted via phosphorylation and dephosphorylation similarly to the regulation of the activity of the PDHc. The phosphorylation of BCKD inhibits the enzyme, whereas, dephosphorylation activates it.
Phosphorylation of BCKD is catalyzed by the kinase, branched chain keto acid dehydrogenase kinase [commonly referred to as BDK; originally identified as 3-methyl-2-oxobutanoate dehydrogenase (lipoamide) kinase] which is encoded by the BCKDK gene. The BCKDK gene is located on chromosome 16p11.2 and is composed of 13 exons that generate three alternatively spliced mRNAs, each of which encode a distinct protein isoform.
The activity of BCKD kinase is itself regulated by phosphorylation, being phosphorylated on Tyr 246 (Y246) by the SRC tyrosine kinase. Phosphorylation of BCKDK by SRC results in repression of the kinase activity of BCKDK. This effect of SRC leads to increased metabolism of BCAA, an effect that is associated with numerous cancers.
Loss-of-function mutations in the BCKDK gene are associated with a phenotype that is characterized by persistently reduced levels of BCAA in body fluids, developmental delay, microcephaly, and neurobehavioral abnormalities. Conversely, gain-of-function mutations in the BCKDK gene are associated with symptoms reflective of a mild form of maple syrup urine disease, MSUD.
The dephosphorylation of BCKD is catalyzed by the phosphatase identified as protein phosphatase, Mg2+/Mn2+ dependent 1K (also known as PP2Cm) which is encoded by the PPM1K gene. The PPM1K gene is located on chromosome 4q22.1 and is composed of 11 exons that encode a 372 amino acid protein.
Both the BCKDK and PPMK1 gene encoded proteins are localized to the mitochondria as is the BCKD complex. Both the BCKD kinase and phosphatase are tightly associated with the E2 subunit of the BCKD complex. Phosphorylation and dephosphorylation of BCKD occurs on a Ser residue of the α-subunits of the E1 heterotetrameric complex.
Byproducts of Branched-Chain Amino Acid Catabolism
The catabolism of leucine gives rise to acetyl-CoA and acetoacetyl-CoA, and therefore, leucine is classified as strictly ketogenic. During the catabolism of leucine, the 3-methylglutaconyl-CoA is converted to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) via the action of the bifunctional enzyme, 3-methylglutaconyl-CoA hydratase. The 3-methylglutaconyl-CoA hydratase enzyme possesses both RNA-binding and hydratase activities accounting for the name of the gene encoding this enzyme, AU RNA-binding methylglutaconyl-CoA hydratase (AUH). The AUH gene is located on chromosome 9q22.31 and is composed of 16 exons that generate five alternatively spliced mRNAs that collectively encode four distinct protein isoforms.
The HMG-CoA derived from the catabolism of leucine can be transported to the cytosol where is can contribute to the synthesis of isoprenoid compounds and cholesterol. Conversion of the mitochondrial HMG-CoA, derived from leucine catabolism, to acetoacetyl-CoA and acetoacetate is catalyzed by HMG-CoA lyase (encoded by the HMGCL gene). This is the same enzyme involved in the synthesis of acetoacetate during ketone body synthesis in the liver.
The principal catabolic by-product from valine is propionyl-CoA, the glucogenic precursor of succinyl-CoA. Isoleucine catabolism terminates with production of acetyl-CoA and propionyl-CoA; thus isoleucine is both glucogenic and ketogenic. As pointed out above for the catabolism of methionine, the resulting propionyl-CoA is converted, via a mitochondrially-localized, three reaction, ATP-dependent pathway, to succinyl-CoA. The succinyl-CoA can then enter the TCA cycle for further oxidation or diverted, via malate, into the gluconeogenesis pathway. The enzymes required for this conversion are propionyl-CoA carboxylase, methylmalonyl-CoA epimerase, and the B12-dependent enzyme, methylmalonyl-CoA mutase. This propionyl-CoA conversion pathway is often remembered by the mnemonic as the VOMIT pathway, where V stands for valine, O for odd-chain fatty acids, M for methionine, I for isoleucine, and T for threonine.
Propionyl-CoA carboxylase functions as a heterododecameric enzyme (subunit composition: α6β6) and the two different subunits are encoded by the PCCA and PCCB genes, respectively. The PCCA gene is located on chromosome 13q32.3 and is composed of 32 exons that generate eleven alternatively spliced mRNAs, each of which encode a unique protein isoform. The PCCB gene is located on chromosome 3q22.3 and is composed of 17 exons that generate two alternatively spliced mRNAs encoding proteins of 539 amino acids (isoform 1) and 559 amino acids (isoform 2). Mutations in either the PCCA or PCCB gene result in the organic acidemia, propionic acidemia.
Methylmalonyl-CoA epimerase is encoded by the MCEE gene located on chromosome 2p13.3 and is composed of 4 exons that encode a 176 amino acid precursor protein.
Methylmalonyl-CoA mutase is encoded by the MMUT gene located on chromosome 6p12.3 and is composed of 13 exons that encode a precursor protein of 750 amino acids. Mutations in the MMUT gene are one cause of the methylmalonic acidemias.
Disorders of Branched-Chain Amino Acid Metabolism
Details of the various diseases resulting from mutations in the enzymes of branched-chain amino acid catabolism are presented in the Branched-Chain Amino Acid Metabolism Disorders page as well as in specific pages for individual diseases, for example Maple Syrup Urine Disease, MSUD.
Leucine Signaling and Metabolic Regulation
Numerous studies have shown that diets high in protein increase fatty acid oxidation and overall energy expenditure and thus, promote weight loss. In addition, high protein diets are known to improve glucose homeostasis and therefore, can have a positive impact on insulin sensitivity and diabetes. In animal models of obesity and diabetes, increasing the protein to carbohydrate ratio in a high-fat diet results in a delay in the development of obesity while simultaneously improving glucose tolerance. In studies on obese humans, subjects that consumed a milk whey-protein-enriched diet exhibited demonstrable improvement in fatty liver symptoms (hepatic steatosis) and reduced plasma lipid profiles. The composition of whey protein is approximately 65% β-lactoglobulin, 25% α-lactalbumin, and 8% serum albumin. In studies examining the effects of dietary protein on energy expenditure, appetite suppression, and weight loss, whey protein is the more beneficial source when comparing the effects of consumption of whey proteins, soy proteins, or casein.
Whey proteins are enriched in the branched-chain amino acids (BCAA) leucine, isoleucine, and valine and are thus, excellent sources of energy production in skeletal muscle as well as serving as building blocks for muscle protein synthesis. Leucine has been proposed to be the primary mediator of the metabolic changes that occur when consuming a high protein diet. At the molecular level, leucine has been shown to activate the metabolic regulatory kinase known as mammalian target of rapamycin, mTOR. For more information on the activities and regulation of mTOR go to the Insulin Function, Insulin Resistance, and Food Intake Control of Secretion page or to the Protein Synthesis (Translation): Processes and Regulation page.
Activation of skeletal muscle mTOR results in increased protein synthesis and thus, increased energy expenditure. Hypothalamic mTOR activation is also involved in the regulation of feeding behaviors. Of note is the fact that direct injection of leucine into the hypothalamus results in increased mTOR signaling leading to decreased feeding behavior and body weight. This effect is unique to leucine, as direct injection of valine, another BCAA, does not result in hypothalamic mTOR activation nor reductions in food intake or body weight.
The effects of leucine supplementation, on the above described parameters, are not as pronounced as the effects observed when consuming a high-protein diet. This suggests that additional factors are likely involved in the effects of high-protein diets. One of these factors may be that consuming a high protein diet is associated with a reduction in total carbohydrate intake. The reduced carbohydrate intake would thus, be associated with a reduction in hepatic lipogenesis and an increase in adipose tissue lipolysis. Although leucine alone is not as effective as high-protein diets at reducing body weight and food intake it cannot be discounted as an important dietary supplement.
However, it must be pointed out that some controversy surrounds the role of high protein consumption, and in particular leucine intake, in overall metabolic homeostasis. This is due to the fact that some studies in laboratory animals have shown that leucine supplementation results in insulin resistance. This latter effect would certainly lead to an increased likelihood for development of type 2 diabetes.
In the typical Western diet consisting of high dairy and meat, the role of leucine in the pathogenesis of type 2 diabetes is suggested by the consequent over activation of mTOR. With respect to diabetes, activation of mTOR results in phosphorylation and activation of the kinase, p70S6K, which in turn phosphorylates the insulin receptor substrate 1 (IRS-1). Phosphorylation of IRS-1 by p70S6K results in reduced insulin-mediated signaling via the insulin receptor, thereby, increasing the metabolic burden on pancreatic β-cells. In addition, increased mTOR activation leads to adipogenesis which can lead to obesity-mediated insulin resistance.
Most of the work in laboratory animals demonstrates that the positive effects of increased leucine intake or high-protein diets are most pronounced when the animals are also consuming a high-fat diet. Therefore, it is suggestive that there are definite benefits for overweight and obese individuals to increase their intake of leucine and/or total protein as a means for appetite and weight control. However, although high-protein diets or leucine supplementation are important considerations in a healthy diet, the total amount consumed must be taken into account so as not to lead to excess mTOR activation.
Phenylalanine and Tyrosine Catabolism
Phenylalanine normally has only two fates: incorporation into polypeptide chains, and hydroxylation to tyrosine via the tetrahydrobiopterin-requiring phenylalanine hydroxylase (PAH) reaction. Thus, phenylalanine catabolism always ensues in the pathway of tyrosine biosynthesis followed by tyrosine catabolism. Tyrosine is equally important for protein biosynthesis as well as an intermediate in the biosynthesis of the catecholamines: dopamine, norepinephrine and epinephrine.
The pathway of tyrosine degradation involves conversion to fumarate and acetoacetate, allowing phenylalanine and tyrosine to be classified as both glucogenic and ketogenic. The catabolism of tyrosine involves five reactions, four of which have been shown to be associated with inborn errors in metabolism and three of these result in clinically significant disorders.
The first reaction of tyrosine catabolism involves the nuclear genome encoded mitochondrial enzyme tyrosine aminotransferase and generates the corresponding ketoacid, p-hydroxyphenylpyruvic acid. Like most aminotransferase reaction, tyrosine aminotransferase utilizes 2-oxoglutarate (α-ketoglutarate) as the amino acceptor with the consequent generation of glutamate. Tyrosine aminotransferase is encoded by the TAT gene that is located on chromosome 16q22.2 and is composed of 12 exons that generate a protein of 454 amino acids.
The second reaction of tyrosine catabolism is catalyzed by 4-hydroxyphenylpyruvate dioxygenase which is encoded by the HPD gene located on chromosome 12q24.31 which is composed of 17 exons that generate two alternatively spliced mRNAs encoding proteins of 393 amino acids (isoform 1) and 354 amino acids (isoform 2).
The product of the HPD reaction is homogentisic acid (homogentisate). Homogentisate is oxidized by the second dioxygenase enzyme of tyrosine catabolism, homogentisate oxidase. Homogentisate oxidase is encoded by the homogentisate 1,2-dioxygenase gene, HGD. The HGD gene is located on chromosome 3q13.33 and is composed of 16 exons that encode a protein of 445 amino acids.
Oxidation of homogentisate yields 4-maleylacetoacetate which is isomerized to 4-fumarylacetoacetate by the enzyme glutathione S-transferase zeta (ζ) 1 which is encoded by the GSTZ1 gene. Glutathione S-transferase zeta 1 was formerly called 4-maleylacetoacetate isomerase or maleylacetoacetate cis–trans-isomerase. The GSTZ1 gene is located on chromosome 14q24.3 and is composed of 9 exons that generate four alternatively spliced mRNAs, each of which encode a distinct protein isoform. One GSTZ1 encoded protein lacks the glutathione binding site and is, therefore, unlikely to exhibit the enzymatic activity of the other three isoforms.
Fumarylacetoacetate is hydrolyzed to fumarate and acetoacetate by the enzyme fumarylacetoacetate hydrolase which is encoded by the FAH gene located on chromosome 15q25.1 and is composed of 15 exons that generate three alternatively spliced mRNAs, each of which encode the same 419 amino acid protein.
The fumarate end product of tyrosine catabolism feeds directly into the TCA cycle for further oxidation. The acetoacetate is activated to acetoacetyl-CoA via the action of the mitochondrial ketone body utilization enzyme, succinyl-CoA:3-oxoacid-CoA transferase (SCOT) which is encoded by the OXCT1 (3-oxoacid-CoA transferase 1) gene. Acetoacetate can also be activated in the cytosol by the cytosolic enzyme, acetoacetyl-CoA synthetase (AACS).
Disorders Associated with Defective Tyrosine Metabolism
Defects in three of the enzymes of tyrosine catabolism result in increased concentrations of tyrosine and metabolites in the blood and urine. These disorders are referred to as the tyrosinemias and are also often referred to as the hypertyrosinemias. Because of the involvement of mutations in three distinct genes in the etiology of tyrosinemia, there are three distinct forms identified as tyrosinemia type 1, type 2, and type 3.
Mutations in the FAH gene result in life-threatening disorder, tyrosinemia type 1 (TYRSN1). TYRSN1 is an autosomal recessive disorder characterized by hypertyrosinemia and progressive liver disease. This disorder also leads to a secondary renal tubular dysfunction resulting in hypophosphatemic rickets. Defects in the FAH gene result in the accumulation of the upstream tyrosine metabolites, maleylacetoacetate and fumarylacetoacetate, as well as their metabolic by-products succinylacetone and succinylacetoacetate. The compounds are toxic to cells, particularly liver and kidney, explaining the pathology associated with this disease. Succinylacetone is also a potent inhibitor of δ-aminolevulinic acid dehydratase (also called porphobilinogen synthase) leading to accumulation of δ-aminolevulinic acid (ALA). ALA is neurotoxic and thought to be the cause of the acute porphyria-like pathology in this TYRSN1.
Inherited mutations in the TAT gene lead to tyrosinemia type 2 (TYRSN2; also called Richner-Hanhart syndrome) which, as the name implies, is associated with elevated tyrosine levels in the blood and, consequently the urine. This form of tyrosinemia is associated with intellectual impairment, painful corneal eruptions, photophobia, keratitis, and painful palmoplantar hyperkeratosis.
Mutations in the HPD gene result in tyrosinemia type 3 (TYRSN3). TYRSN3 is an autosomal recessive disease that, in addition to hypertyrosinemia, is associated with mild intellectual impairment and/or convulsions but these patients do not display hepatic damage as is characteristic of tyrosinemia type 1.
The first inborn error in metabolism ever recognized, alkaptonuria, was demonstrated to be the result of a defect in phenylalanine and tyrosine catabolism. Alkaptonuria is caused by defective homogentisic acid oxidase which is the third enzyme in the tyrosine catabolic pathway. Homogentisic acid accumulation is relatively innocuous, causing urine to darken on exposure to air, but no life-threatening effects accompany the disease. The only untoward consequence of alkaptonuria is ochronosis (bluish-black discoloration of the tissues) and arthritis of indeterminate etiology.
Lysine Catabolism
Lysine, Arginine, and Ornithine Transporters
Like all amino acids, catabolism of lysine can initiate from uptake of dietary lysine or from the breakdown of intracellular protein. Intestinal uptake of lysine involves specific transporter proteins. In most tissues, cationic amino acids are transported principally by a Na+-independent system, specific for L-isomers of lysine, arginine, and ornithine. This transport system is known as the y+ system and these transporters are members of the SLC7 family of membrane transporters where this Na+-independent transporter is encoded by the SLC7A1 gene.
There are actually at least three transporter mechanisms for lysine transport. One is the aforementioned y+ system that functions as a uniporter. The other two lysine transporters are Na+-dependent antiporter systems that are heterodimeric transporters composed of a heavy chain and a light chain forming transporters of the y+L system.
The common heavy chain of these two different transporters is encoded by the SLC3A2 gene (often referred to as 4F2hc for 4F2 cell-surface antigen heavy chain). The light chains are encoded by the SLC7A6 and SLC7A7 genes which encode the y+LAT2 and y+LAT1 proteins, respectively. Once taken up by the intestines, dietary lysine can be incorporated into protein or catabolized.
The SLC7A1 encoded transporter is the primary transporter for the cellular uptake of arginine whereas the antiporter composed of the SLC7A6 encoded protein is the major arginine efflux transporter for transport of arginine out of cells.
Lysine Catabolism via the Saccharopine Pathway
There are several, at least three, pathways for lysine catabolism but the primary pathway utilized within the liver of humans is one that begins with the formation of an adduct between lysine and 2-oxoglutarate (α-ketoglutarate) called saccharopine. Lysine catabolism is unusual in the way that the ε-amino group is transferred to 2-oxoglutarate and into the general nitrogen pool. The reaction is a transamination in which the ε-amino group is transferred to the α-keto carbon of 2-oxoglutarate (α-ketoglutarate) forming the metabolite, saccharopine. Unlike the majority of transamination reactions, the enzyme catalyzing the formation of saccharopine does not employ pyridoxal phosphate as a cofactor.
The formation of saccharopine and its hydrolysis to α-aminoadipic-6-semialdehyde is catalyzed by the bifunctional enzyme α-aminoadipic semialdehyde synthase. This reaction results in the amino nitrogen remaining with the α-carbon of 2-oxoglutarate, producing glutamate and α-aminoadipic-6-semialdehyde. Because this transamination reaction is not reversible, lysine is an essential amino acid. Mammalian α-aminoadipic semialdehyde synthase is an NADPH-dependent mitochondria-localized enzyme encoded by the AASS gene.
The AASS gene is located on chromosome 7q31.32 and is composed of 25 exons encoding a mitochondrially localized protein of 926 amino acids. The N-terminal half of the AASS protein harbors the lysine:2-oxoglutarate reductase activity and the C-terminal half harbors the saccharopine dehydrogenase activity.
The α-aminoadipic-6-semialdehyde is catabolized to α-aminoadipic acid via the action of the NAD+-dependent enzyme, aldehyde dehydrogenase 7 family member A1 (encoded by the ALDH7A1 gene), originally identified as α-aminoadipic semialdehyde dehydrogenase (AASA dehydrogenase). This enzyme is also known as antiquitin (ATQ1).
The ALDH7A1 gene is located on chromosome 5q23.2 and is composed of 18 exons that generate three alternatively spliced mRNAs. Two of these ALDH7A1 derived mRNAs each encode two proteins through the use of alternative in-frame translation initiation codons. One of the resultant proteins is localized to the mitochondria the other to the cytosol. It is the mitochondrial enzyme that is involved in lysine catabolism.
The α-aminoadipic acid is then deaminated to 2-oxoadipic acid (2-OAA; also called α-ketoadipic acid or 2-ketoadipic acid) via the transaminase, aminoadipate aminotransferase (encoded by the AADAT gene). Aminoadipate aminotransferase is highly similar to the rodent kynurenine aminotransferase II (KATII) and the AADAT encoded enzyme is also involved in the conversion of kynurenine to kynurenic acid in the tryptophan catabolism pathway.
The AADAT gene is located on chromosome 4q33 and is composed of 18 exons that generate four alternatively spliced mRNAs that collectively encode two proteins isoforms of 429 amino acids (isoform a) and 425 amino acids (isoform b)
The 2-oxoadipic acid then undergoes oxidative decarboxylation to glutaryl-CoA via the actions of the 2-oxoadipate dehydrogenase complex. This enzyme complex is composed of the E1 component protein encoded by the DHTKD1 (dehydrogenase E1 and transketolase domain containing 1) gene and shares the E2 (dihydrolipoamide S-succinyltransferase: DLST) and E3 (dihydrolipoamide dehydrogenase: DLD) components of the 2-oxoglutarate complex of the TCA cycle (commonly called α-ketoglutarate dehydrogenase).
The DHTKD1 gene is located on chromosome 10p14 and is composed of 17 exons that encode a 919 amino acid protein.
Glutaryl-CoA is oxidized to crotonyl-CoA by the FAD-dependent enzyme, glutaryl-CoA dehydrogenase (GCDH). Glutaryl-CoA dehydrogenase is a member of the acyl-CoA dehydrogenase (ACAD) family of enzymes that includes members of the mitochondrial fatty acid β-oxidation pathway and as such the enzyme is also known as acyl-CoA dehydrogenase 5 (ACAD5). Mutations in the GCDH gene result in the disorder identified as glutaric aciduria/acidemia type 1.
The crotonyl-CoA is converted to β-hydroxybutyryl-CoA and β-hydroxybutyryl-CoA is converted to acetoacetyl-CoA, the last reaction being catalyzed by hydroxysteroid 17-beta dehydrogenase 10 (encoded by the HSD17B10 gene) which is also known as 3-hydroxybutyryl-CoA dehydrogenase.
Crotonyl-CoA can also serve as the substrate for the post-translational modification of lysine residues, a process termed protein crotonylation.
Disorders Associated with Defective Lysine Catabolism
Genetic deficiencies in either of the first two reactions of the saccharopine pathway of lysine catabolism result in hyperlysinemia type 1. Hyperlysinemia type 1 is an autosomal recessive disorder exhibiting variable phenotype. Since these two reactions are catalyzed by the bifunctional enzyme, AASS, defects can be found in either the N-terminal lysine:2-oxoglutarate reductase activity or the C-terminal saccharopine dehydrogenase activity. These deficiencies are observed in individuals who excrete large quantities of urinary lysine, pipecolic acid, and some saccharopine. The most severe form of hyperlysinemia type 1 manifests in infancy with nonspecific seizures, hypotonia, or mildly delayed psychomotor development. Many individuals are asymptomatic and the hyperlysinemia is considered a benign condition in these individuals.
Other serious disorders associated with lysine metabolism are due to failure of the transport systems for lysine and the other dibasic amino acids across the intestinal wall. Lysine is essential for protein synthesis; and deficiencies of its transport into the body can cause seriously diminished levels of protein synthesis. Probably more significant however, is the fact that arginine is transported by the same dibasic amino acid transporter, and resulting arginine deficiencies limit the quantity of ornithine available for the urea cycle. The result is severe hyperammonemia after a meal rich in protein. The addition of citrulline to the diet prevents the hyperammonemia.
Minor Pathways of Lysine Catabolism
One of the minor pathways of lysine catabolism is the pipecolic acid pathway. L-pipecolic acid (2-carboxypiperdine) was known to be a widely distributed non-protein amino acid in plants and to be derived from lysine catabolism in mammals. Originally it was thought that the degradation of lysine to pipecolate was the major catabolic pathway for this amino acid. Although it is now known that this product of lysine catabolism reflects a minor catabolic pathway, there is clinical significance to this pathway.
In addition, lysine catabolism via the pipecolic acid pathway may play a significant role in the central nervous system. Lysine catabolism via this pathway involves conversion to α-keto-ε-amino-caproic acid and then to pipecolic acid. Pipecolic acid then is ultimately converted to α-aminoadipic semialdehyde which is also a product of the saccharopine pathway of lysine catabolism.
Formation of α-aminoadipic semialdehyde from pipecolic acid is catalyzed by the dehydrogenase encoded by the aldehyde dehydrogenase 7 family member A1 gene (ALDH7A1). As indicated above this enzyme is also known as α-aminoadipic semialdehyde dehydrogenase (AASA dehydrogenase) and is also known as antiquitin (ATQ1).
Carnitine Synthesis from Lysine
Lysine is also important as a precursor for the synthesis of carnitine (γ-trimethyl-β-hydroxybutyrobetaine; also identified as β-hydroxy-γ-N-trimethylamino-butyric acid), required for the transport of fatty acids into the mitochondria for oxidation. Free lysine does not serve as the precursor for this reaction, rather the modified lysine (6-N-trimethyllysine), found in certain proteins, is the precursor.
Protein lysine methylation is catalyzed by one of a family of 27 lysine methyltransferases (KMT) expressed in humans. Some of the most critical proteins that are methylated on lysine residues are the histone proteins in chromatin, the consequences of which are regulation of gene expression. The methyl donor for KMT proteins is S-adenosylmethionine (AdoMet of SAM).
Hydrolysis of proteins containing trimethyllysine provides the substrate for the subsequent conversion to carnitine. Once trimethyllysine is released from proteins within the lysosomal compartment, the overall pathway to carnitine synthesis requires four separate reactions.
Most human tissues can convert trimethyllysines to γ-butyrobetaine (the compound prior to carnitine) but only the liver, kidney, brain, and testes express the γ-butyrobetaine dioxygenase enzyme (also called γ-butyrobetaine 2-oxoglutarate dioxygenase or γ-butyrobetaine hydroxylase) that hydroxylates γ-butyrobetaine forming carnitine. The γ-butyrobetaine dioxygenase enzyme as well as the first enzyme of carnitine synthesis (trimethyllysine hydroxylase, encoded by the TMLHE gene) are members of the large family of 2-oxoglutarate and ferrous (Fe2+) iron-dependent dioxygenases. Although the liver, kidney, brain, and testes can synthesize carnitine, only the liver and the kidneys can transport carnitine to the blood.
The TMLHE gene is located on the X chromosome (Xq28) and is composed of 13 exons that generate two alternatively spliced mRNAs that encode precursor proteins of 421 amino acids (isoform 1) and 376 amino acids (isoform 2).
The genes encoding the serine hydroxymethyltransferases, suspected to catalyze the second reaction of carnitine synthesis, are described in the section above on Serine Biosynthesis.
The third step in carnitine synthesis is catalyzed by aldehyde dehydrogenase 9 family, member A1 (ALDH9A1). The ALDH9A1 encoded enzyme was formerly known as 4-N-trimethylaminobutyraldehyde dehydrogenase (TMABADH).
The ALDH9A1 gene is located on chromosome 1q24.1 and is composed of 11 exons that generate two alternatively spliced mRNAs that encode proteins of 518 amino acids (isoform 1) and 424 amino acids (isoform 2).
The gene encoding γ-butyrobetaine dioxygenase is identified as BBOX1. The BBOX1 gene is located on chromosome 11p14.2 and is composed of 11 exons that generate five alternatively spliced mRNAs, all of which encode the same 387 amino acid protein.
Functions of Carnitine
The major function of carnitine is in the activation of long-chain fatty acids, generating fatty acyl-carnitines, which is required for the transport of long-chain fatty acids into the mitochondria for oxidation. However, carnitine is also involved in the metabolism of ketones, and the catabolism of the branched-chain amino acids. Carnitine is also important for the removal of fatty acyl-CoA metabolites so as to reduce the likelihood for the accumulation of toxic intermediates.
The major carnitine derivatives, that are not the long-chain fatty acylcarnitines involved in fatty acid acid oxidation, are acetyl-carnitine (acetyl-L-carnitine, ALC), propionyl-L-carnitine (PLC), and isovaleryl-L-carnitine (iso-VLC). Both acetyl-carnitine and propionyl-carnitine have been shown to improve insulin sensitivity (reduce insulin resistance) most likely as a result of inhibiting the transport of long-chain fatty acids into cells. Several studies have also demonstrated that propionyl-carnitine is able to lessen the development of atherosclerosis and lessen endothelial cell dysfunction. Acetyl-carnitine also crosses the blood brain barrier and in the brain modifies CNS acetylcholine production as well as improving energy production by neurons.
In addition to these three major carnitine derivates, human cells produce butyryl-L-carnitine, hydroxybutyryl-L-carnitine, hexanoyl-L-carnitine, ortanoyl-L-carnitine, decanoyl-L-carnitine, palmitoyl-L-carnitine, stearoyl-L-carnitine, and acetoacetyl-L-carnitine.
Histidine Catabolism
Histidine catabolism begins with release of the α-amino group catalyzed by histidine ammonia-lyase (also called histidase), introducing a double bond into the molecule. As a result, the deaminated product, urocanate, is not the usual α-keto acid associated with loss of α-amino nitrogens.
Histidine ammonia-lyase is encoded by the HAL gene. The HAL gene is located on chromosome 12q23.1 and is composed of 22 exons generating three alternatively spliced mRNAs, each of which encode a distinct protein isoform. The end product of histidine catabolism is glutamate, making histidine one of the glucogenic amino acids.
Another key feature of histidine catabolism is that it serves as a source of ring nitrogen to combine with tetrahydrofolate (THF), producing the 1-carbon THF intermediate known as N5-formimino-THF. The latter reaction is one of two routes to N5-formimino-THF.
Urocanate is converted to 4-imidazolone-5-propionate via the action of urocanate hydratase 1 (encoded by the UROC1 gene). The UROC1 gene is located on chromosome 3q21.3 and is composed of 21 exons that generate two alternatively spliced mRNAs encoding proteins of 676 amino acids (isoform 1) and 736 amino acids (isoform 2).
The 4-imidazolone-5-propionate is then converted to N-formimidoyl-L-glutamate via the action of an imidazolone propionase activity. The imidazolone propionase activity is encoded by the amidohydrolase domain-containing 1 gene (symbol: AMDHD1). The AMDHD1 gene is located on chromosome 12q23.1 and is composed of 9 exons that encode a 426 amino acid protein.
The bifunctional enzyme, formimidoyltransferase cyclodeaminase (also called glutamate formiminotransferase), then transfers the formimino group from N-formimidoyl-L-glutamate to THF yielding N5-formimino-THF (5-fominino-THF) and glutamate. Formimidoyltransferase cyclodeaminase functions as a homooctameric complex. The 5-formimino-THF is then converted to N5,N10-methenylene-THF (5,10-methenylene-THF) via the cyclodeaminase activity of the complex with glutamate and ammonium ion as the other products.
Formimidoyltransferase cyclodeaminase is encode by the FTCD gene. The FTCD gene is located on chromosome 21q22.3 and is composed of 16 exons that generate three alternatively spliced mRNAs, two of which encode the same 541 amino acid protein (isoform A) and the other encodes a protein of 572 amino acids (isoform C).
Disorders Associated with Defective Histidine Catabolism
The principal genetic deficiencies associated with histidine metabolism are associated with mutations in the HAL, UROC1, and FTCD gene. Mutations in the HAL gene result in histidinemia which is relatively benign. The disease, which is of relatively high incidence (1 in 10,000), is most easily detected by the absence of urocanate from skin and sweat, where it is normally found in relative abundance.
UROC1 mutations have only been reported in a few individuals who inherited compound heterozygous missense mutations. These individuals exhibited intellectual impairment, short stature, and episodic aggressiveness and affection-seeking behaviors.
Inherited defects in the FTCD gene represent the second most common cause of inborn errors in folate metabolism. Potential for severe intellectual impairment is the main clinical feature associated with FTCD deficiency. Patients with FTCD defects will also present with megaloblastic anemia due to the role of folate derivatives in the synthesis of purine and thymine nucleotides.
Histamine Synthesis and Functions
Decarboxylation of histidine in the intestine by bacteria gives rise to histamine. Similarly, histamine is synthesized in numerous tissues by the decarboxylation of histidine catalyzed by histidine decarboxylase which is a pyridoxal phosphate-requiring (B6) enzyme.
The histidine decarboxylase is encoded y the HDC gene. The HDC gene is located on chromosome 15q21.2 and is composed of 14 exons that generate two alternatively spliced mRNAs that encode proteins of 662 amino acids (isoform 1) and 629 amino acids (isoform 2).
Histamine exerts multiple activities (see the Biochemistry of Nerve Transmission page) but the two most significant are its roles in immunologic response to antigen and inducing the release of hydrogen ion (H+) by parietal cells of the stomach to generate the HCl necessary for gastric digestion. The largest amount of histamine produced in the body is that made by mast cells and basophils. If these cells are sensitized by the presentation of IgE on their surfaces they will degranulate in response to antigen binding, releasing histamine into the circulation. The response to this release is bronchoconstriction and vasodilation which are the general symptoms associated with asthma and various allergic reactions.
Within the gastrointestinal system, histamine is produced in the enterochromaffin-like cells of the stomach. When histamine is released from these cells, in response to vagal nerve stimulation, it binds to specific receptors on parietal cells triggering the mobilization of the proton pump of the parietal cell to the apical membrane allowing for increased H+ transport into the lumen of the stomach.
All of the actions of histamine are the result of binding to one of four cell surface receptors (H1R–H4R), all of which are G-protein coupled receptors (GPCR). The H1 receptor is coupled to a Gq-type G-protein and is responsible for triggering bronchoconstriction and vasodilation. The H2 receptor is a Gs-type G-protein and is present on parietal cells and vascular smooth muscle cells.
Tryptophan Catabolism
The major tryptophan catabolic pathway in humans results in the formation of acetoacetyl-CoA. This pathway is referred to as the kynurenine pathway and occurs predominantly in the liver. However, it is important to note that overall metabolism of tryptophan includes not just its catabolism but also its use in the synthesis of serotonin and melatonin, the synthesis of indole derivatives such as the trace amine, tryptamine, and the synthesis of NAD+.
The first enzyme of the kynurenine pathway of tryptophan catabolism in the liver opens the indole ring. This enzyme is tryptophan 2,3-dioxygenase which is encoded by the TDO2 gene. The product of the TDO2 reaction is N-formyl-kynurenine.
The TDO2 gene is located on chromosome 4q32.1 and is composed of 12 exons that encode a protein of 406 amino acids. Expression of the TDO2 gene is very nearly exclusive to the liver. The TDO2 gene is highly inducible, leading to increasing concentrations of the enzyme, of up to 10-fold, on a diet high in tryptophan.
Tryptophan catabolism can also be initiated by the enzyme indoleamine 2,3-dioxygenase 1 encoded by the IDO1 gene. The activity of the IDO1 enzyme also results in the generation of N-formyl-kynurenine.
The IDO1 gene is located on chromosome 8p11.21 and is composed of 10 exons that encode a protein of 403 amino acids. Unlike the TDO2 gene, whose expression is restricted to the liver, the IDO1 gene is expressed in numerous tissues but at levels much lower than the level of liver expression of the TDO2 gene. In addition to tryptophan, IDO1 can metabolize 5-hydroxytryptophan, tryptamine, and serotonin.
N-formyl-kynurenine is then converted to kynurenine by the enzyme, arylformamidase (also called kynurenine formamidase), encoded by the AFMID gene. The AFMID gene is located on chromosome 17q25.3 and is composed of 12 exons that generate two alternatively spliced mRNAs that generate 11 alternatively spliced mRNAs, each of which encode distinct protein isoforms.
Kynurenine is the first key branch point intermediate in the catabolic pathway leading to three distinct catabolic fates for tryptophan. These branches involve conversion of kynurenine to anthranilic acid, to kynurenic acid, or to 3-hydroxyanthranilate. The latter compound can be fully catabolized or serve as the precursor for NAD+ synthesis.
Kynurenine can undergo deamination to kynurenic acid in a transamination reaction. The transamination reaction can be catalyzed by several different transaminases. One of these enzymes is the human homolog of mouse kynurenine aminotransferase II, yielding kynurenic acid. The human kynurenine aminotransferase II (KAT II or KAT2) enzyme is called aminoadipate aminotransferase which is encoded by the AADAT gene. The enzyme encoded by the AADAT gene has two aminotransferase activities, one of which is utilized in the saccharopine pathway of lysine catabolism, the other is used in the transamination of kynurenine. Another kynurenine transaminase is cysteine conjugate-beta lyase 2 encoded by the CCBL2 gene. The CCBL2 encoded protein was formerly known as kynurenine aminotransferase III (KAT III or KAT3). Another related enzyme, that was once called kynurenine aminotransferase I (KAT I or KAT1), is cysteine conjugate-beta lyase encoded by the CCBL1 gene. The CCBL1 encoded protein is a cytosolic enzyme that, as its name implies, metabolizes cysteine conjugates.
Conversion of kynurenine to anthranilic acid involves the enzyme, kynureninase. Kynureninase is a pyridoxal phosphate (vitamin B6)-requiring enzyme encoded by the KNYU gene. The KYNU gene is located on chromosome 2q22.2 and is composed of 18 exons that generate three alternatively spliced mRNAs that collectively encode two distinct protein isoform. Expression of the KYNU gene is highest in the liver which is to be expected since the majority of tryptophan catabolism occurs in this organ.
Hydroxylation of kynurenine via the action of kynurenine hydroxylase yields 3-hydroxykynurenine. Kynurenine hydroxylase is correctly called kynurenine 3-monooxygenase and it is encoded by the KMO gene. The KMO gene is located on chromosome 1q43 and is composed of 15 exons that generate two alternatively spliced mRNA, both of which encode distinct protein isoforms. 3-Hydroxykynurenine is then converted to 3-hydroxyanthranilate via the action of kynureninase.
Catabolism of α-Amino-β-Carboxymuconate-ε-Semialdehyde: ACMS
The α-amino-β-carboxymuconate-ε-semialdehyde (ACMS) product of the HAAO catalyzed reaction can be decarboxylated to α-aminomuconate-ε-semialdehyde (AMS) by the enzyme, α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (encoded by the ACMSD gene).
The ACMSD gene is located on chromosome 2q21.3 and is composed of 13 exons that generate two alternatively spliced mRNAs. Only one mRNA encodes a functional protein as the other is likely degraded via the nonsense-mediated decay (NMD) pathway. The functional ACMSD encoded protein (isoform 1) is 336 amino acids.
AMS is ultimately converted to 2-oxoadipic acid (2-OAA; also called α-ketoadipic acid or 2-ketoadipic acid) through a series of two reactions and and then to glutaryl-CoA. The lysine catabolic pathway merges with the tryptophan catabolic pathway with the production of 2-OAA and thus, the remainder of both pathways are the same. The 2-oxoadipic acid undergoes oxidative decarboxylation to glutaryl-CoA via the actions of the 2-oxoadipate dehydrogenase complex whose E1 subunits are encoded by the DHTKD1 gene. The E2 and E3 subunits of this complex are shared with the 2-oxoglutarate dehydrogenase (α-ketoglutarate dehydrogenase) complex of the TCA cycle.
Glutaryl-CoA is oxidized to crotonyl-CoA by the FAD-dependent enzyme, glutaryl-CoA dehydrogenase (GCDH). Glutaryl-CoA dehydrogenase is a member of the acyl-CoA dehydrogenase (ACAD) family of enzymes that includes members of the mitochondrial fatty acid β-oxidation pathway and as such the enzyme is also known as acyl-CoA dehydrogenase 5 (ACAD5).
Glutaryl-CoA dehydrogenase is encoded by the GCDH gene. The GCDH gene is located on chromosome 19p13.13 and is composed of 12 exons that generate two alternatively spliced mRNAs, both of which encode distinct precursor proteins, isoform a is 438 amino acids and isoform b is 428 amino acids, The isoform b protein is not functional as a glutaryl-CoA dehydrogenase. The isoform a precursor protein undergoes cleavage by mitochondrial processing peptidase complex to form the mature glutaryl-CoA dehydrogenase enzyme. Functional glutaryl-CoA dehydrogenase is a homotetrameric complex. Mutations in the GCDH gene result in the organic acidemia called glutaric acidemia type 1.
As in the lysine catabolic pathway, the crotonyl-CoA derived from tryptophan catabolism is converted to β-hydroxybutyryl-CoA and β-hydroxybutyryl-CoA is converted to acetoacetyl-CoA, the last reaction being catalyzed by hydroxysteroid 17-beta dehydrogenase 10 (encoded by the HSD17B10 gene) which is also known as 3-hydroxybutyryl-CoA dehydrogenase.
Crotonyl-CoA can also serve as the substrate for the post-translational modification of lysine residues, a process termed protein crotonylation.
Tryptophan as Precursor for NAD+
Kynurenine can also undergo a series of catabolic reactions that allow kynurenine to serve as an important intermediate in the pathway for the synthesis of the nicotinamide adenine dinucleotide co-factors, NAD+ and NADP+. In the context of this pathway a potion of the molecule undergoes oxidation to acetoacetyl-CoA.
The conversion of tryptophan to NAD+, which is considered the de novo synthesis pathway, occurs predominantly in the liver. The first reaction in this pathway of kynurenine metabolism is catalyzed by kynurenine 3-monooxygenase (also called kynurenine hydroxylase) forming 3-hydroxykynurenine. Kynurenine 3-monooxygenase is encoded by the KMO gene. The KMO gene is located on chromosome 1q43 and is composed of 15 exons that generate two alternatively spliced mRNAs, both of which encode distinct protein isoforms.
The next step in the pathway is catalyzed by kynureninase and produces 3-hydroxyanthranilic acid and the amino acid alanine. It is the production of alanine that allows tryptophan to be classified among the glucogenic amino acids. As indicated above kynureninase, which is encoded by the KNYU gene, is also used for the conversion of kynurenine to anthranilic acid with the release of alanine. The anthranilic acid subsequently is converted to 3-hydroxyanthranilic acid. This latter two-step reaction is a minor component of overall tryptophan catabolism.
The next step in the pathway is the conversion of 3-hydroxyanthranilic acid to quinolinic acid catalyzed by the enzyme 3-hydroxyanthranilate 3,4-dioxygenase which is encoded by the HAAO gene. The product of the HAAO reaction is α-amino-β-carboxymuconate-ε-semialdehyde (ACMS). ACMS can spontaneously cyclize to quinolinic acid.
The HAAO gene is located on chromosome 2p21 and is composed of 11 exons that encode a 286 amino acid protein. Expression of the HAAO gene is highest in the liver with expression in the small intestine and kidney being the next highest locations.
Quinolinic acid is ultimately converted, via three steps, to the nicotinamide adenine dinucleotide co-factors. The three steps in the conversion of quinolinic acid (quinolinate) to NAD+ first involves the conversion to nicotinate mononucleotide (NAMN), then to nicotinate adenine dinucleotide (NAAD), and then to NAD+.
The first reaction is catalyzed by quinolinate phosphoribosyltransferase which is encoded by the QPRT gene. The QPRT gene is located on chromosome 16p11.2 and is composed of 4 exons that generate three alternatively spliced mRNAs, each of which encode a distinct protein isoform.
The second reaction is catalyzed by one of the nicotinamide nucleotide adenylyltransferase (NMNAT) family enzymes. Humans express three NMNAT encoding genes. The final reaction is catalyzed by NAD synthase (encoded by the NADSYN1 gene). The details of the latter two reactions are described in the Vitamin B3: Metabolism and Functions page.
Clinical Significance of Kynurenine and Kynurenate
The levels of kynurenine have been shown to increase with the aging process as well as in conditions of inflammation. The aging related increases in kynurenine have been shown to be linked to increased frailty and mortality. In organisms such as the nematode (roundworm), Caenorhabditis elegans, and the fruit fly, Drosophila melanogaster, experiments have shown that elevated levels of kynurenine are directly correlated to reduced life span. The mechanism by which kynurenine contributes to these processes is due to the fact that it is an agonist of the aryl hydrocarbon receptor (AHR).
The AHR was originally identified as a protein that bound, and was activated by, the highly toxic chemicals of the dioxin family. Kynurenine was the first endogenous ligand identified as binding to and activating the AHR. In addition to kynurenine the AHR binds additional endogenous ligands such as tryptophan and the tryptophan-derived bioactive trace amine, tryptamine.
There are numerous AHR agonists, both exogenous and endogenous, and their effects upon activation of the receptor can be pathological or beneficial. Naturally occurring plant derived phytochemicals, such as quercetin, exert anti-aging effects via the AHR. In contrast kynurenine exerts pathological effects related to aging. These differences are due to ligand-specific downstream signaling processes. Kynurenine activation of AHR leads to enhancement of oxidative stress and inflammation as a result of increased expression of the cytochrome P450 enzymes encoded by the CYP1A1 and CYP1B1 genes, the interleukin-6 (IL-6) gene, and the transforming growth factor beta 1 (TGFB1) gene. Quercetin on the other hand appears to induce AHR activation of an antioxidant pathway via activation of the basic region leucine zipper (bZip) transcription factor, nuclear factor erythroid 2–related factor 2 (NRF2).
Kynurenic acid has been shown exert protective effects within the central nervous system (CNS) in part by acting as an anti-excitotoxic and anti-convulsive. Conversely, high levels of kynurenic acid have been found in the urine of individuals suffering from schizophrenia. Kynurenic acid has been shown to act as a non-competitive antagonist at the glycine binding site of the NMDA receptor (NMDA: N-methyl-D-aspartate) which is an ionotropic (ligand-gated ion channel) receptor for glutamate. The NMDA receptor is a key component of the glutamatergic neurotransmission system believed to be involved in the pathophysiology of schizophrenia, thus explaining the potential role of kynurenic acid in schizophrenia.
Tryptophan Metabolism and Neurotransmission
Quinolinic acid is also an excitatory neurotoxic compound that functions via its ability to activate the glutamate NMDA receptors. An intermediate in the HAAO catalyzed reaction is α-amino-β-carboxymuconate-ε-semialdehyde (or simply aminocarboxymuconate semialdehyde: ACMS). The ACMS intermediate can be metabolized via the action of the enzyme ACMS decarboxylase encoded by the ACSMD gene.
The ACMSD gene is located on chromosome 2q21.3 and is composed of 13 exons that generate two alternatively splice mRNAs. The isoform 2 mRNA is a likely candidate for nonsense-mediated decay, NMD. The isoform 1 mRNA encodes a protein of 336 amino acids.
The ACMSD catalyzed reaction is important for the prevention of over accumulation of quinolinic acid from ACMS. The product of the ACMSD reaction goes on to be further metabolized to acetoacetyl-CoA, the ultimate end product of complete tryptophan catabolism.
In addition to its role as an amino acid in protein biosynthesis and as a precursor for the synthesis of NAD+, tryptophan also serves as a precursor for the synthesis of the neurotransmitters serotonin and melatonin. These products are discussed in the Amino Acid Derivatives: Neurotransmitters, Nitric Oxide, and More page.