The bulk of ATP used by many cells, to maintain homeostasis, is produced by the re-oxidation of the reduced electron carriers, NADH and FADH2, within the mitochondrial oxidative phosphorylation pathway. A large percentage of these two reduced electron carriers are generated by the oxidation of pyruvate in the TCA cycle. The fate of pyruvate depends on the cell energy charge. In liver, intestine, and kidney when the energy charge is high, pyruvate is directed toward gluconeogenesis. However, when the energy charge is low, pyruvate is preferentially oxidized to CO2 and H2O in the TCA cycle, with generation of 15 equivalents of ATP per pyruvate. The enzymatic activities of the TCA cycle (and of oxidative phosphorylation) are located within the inner mitochondrial matrix and inner mitochondrial membrane.
The transport of pyruvate into the mitochondria involves two distinct sets of transport complexes. The outer mitochondrial membrane transports pyruvate into the intermembrane compartment via the action of a voltage-gated porin complex. Transport of pyruvate across the inner mitochondrial membrane requires the mitochondrial pyruvate carrier (MPC) complex. The MPC is a heterotetrameric complex composed of subunits encoded by the MPC1 and MPC2 genes. The MPC1 gene is located on chromosome 6q27 and is composed of 7 exons that generate two alternatively spliced mRNAs encoding MPC1 isoform 1 (109 amino acids) and MPC1 isoform 2 (66 amino acids). The MPC2 gene is located on chromosome 1q24 and is composed of 7 exons that generate two alternatively spliced mRNAs that both encode the same 127 amino acid protein.back to the top
When transported into the inner mitochondrial matrix, pyruvate encounters two principal metabolizing enzymes: pyruvate carboxylase, PC (a gluconeogenic enzyme) and pyruvate dehydrogenase (PDH), the first enzyme of the PDHc. With a high cell-energy charge, co-enzyme A (CoA) is highly acylated, principally as acetyl-CoA, and able to obligately activate pyruvate carboxylase, directing pyruvate toward gluconeogenesis. When the energy charge is low, CoA is not acylated, therefore, pyruvate carboxylase is inactive, and pyruvate is preferentially metabolized via the PDHc and the TCA cycle to CO2 and H2O. The acetyl-CoA produced by the PDHc enters the TCA cycle and the reduced electron carriers (NADH and FADH2) that are generated during the oxidative reactions can then be used to drive ATP synthesis via oxidative phosphorylation.
The PDHc is comprised of multiple copies of three distinct enzyme activities: pyruvate dehydrogenase (PDH, identified as the E1 component), dihydrolipoamide S-acetyltransferase, (DLAT, identified as the E2 component) and dihydrolipoamide dehydrogenase, (DLD, identified as the E3). The PDH activity (E1) is a heterotetrameric complex composed of two α (alpha) and two β (beta) subunits. The α subunits are primarily encoded by the PDHA1 gene located on chromosome Xp22.1 and is composed of 12 exons that generate four alternatively spliced mRNAs. The predominant PDHA1 derived mRNA (isoform 1) encodes a precursor protein of 390 amino acids. Another PDH α-subunit gene (PDHA2) encodes a testis-specific isoform. The β subunits are encoded by the PDHB gene located on chromosome 3p21.1–p14.2 and is composed of 11 exons that generate two alternatively spliced mRNAs. The DLAT gene is located on chromosome 11q23.1 and is composed of 14 exons that encode a 647 amino acid protein. The DLD gene is located on chromosome 7q31–q32 and is composed of 14 exons that generate four alternatively spliced mRNAs with the largest mRNA encodong a protein of 509 amino acids. The DLD encoded proteins function within the PDHc as homodimeric units. The DLD encoded proteins are also functional components of the 2-oxoglutarate dehydrogenase complex (more commonly known as α-ketoglutarate dehydrogenase) of the TCA cycle described below, and of the branched-chain keto-acid dehydrogenase complex (BCKD, an amino acid metabolism enzyme complex).
In addition to the complexity of the PDHc at the level of the total number of protein subunits (30 subunits of E1, 60 subunits of E2, and 12 subunits of E3), the full activity of the PDHc requires five different vitamin-derived coenzymes: CoA, NAD+, FAD, lipoic acid and thiamine pyrophosphate (TPP). A mnemonic that helps one remember these five required co-factors/coenzymes is Tender (TPP) Loving (lipoic acid) Care (CoA) For (FAD) Nancy (NAD+). Three of the coenzymes of the PDHc are tightly bound to enzymes of the complex (TPP, lipoic acid and FAD) and two are employed as carriers of the products of PDHc activity (CoA and NAD+). Two additional dehydrogenase complexes require these same five coenzymes: the 2-oxoglutarate dehydrogenase complex and the BCKD complex.
The pathway of PDHc-mediated oxidation of pyruvate to acetyl-CoA is diagrammed below:
Flow diagram depicting the overall activity of the PDHc. During the oxidation of pyruvate to acetyl-CoA and CO2, by the PDH activities, the electrons flow from pyruvate to the lipoamide moiety of DLAT, then to the FAD cofactor of DLD and finally to reduction of NAD+ to NADH. The acetyl group is linked to coenzyme A (CoASH) in a high energy thioester bond. The acetyl-CoA then enters the TCA cycle for complete oxidation to CO2 and H2O.
The first enzyme of the PDHc is PDH itself (E1) which oxidatively decarboxylates pyruvate. During the course of the reaction the acetyl group derived from decarboxylation of pyruvate is bound to TPP. The next reaction of the complex is the transfer of the two carbon acetyl group from acetyl-TPP to lipoic acid, the covalently bound coenzyme of DLAT. The transfer of the acetyl group from acyl-lipoamide to CoA results in the formation of 2 sulfhydryl (–SH) groups in lipoate requiring re-oxidation of the disulfide (-S–S-) form to regenerate lipoate as a competent acyl acceptor. The enzyme DLD, with FAD as a cofactor, catalyzes that oxidation reaction. The final activity of the PDHc is the transfer of reducing equivalents from the FADH2 of DLD to NAD+. The fate of the NADH is oxidation via mitochondrial electron transport, to produce approximately three three equivalents of ATP:
The net result of the reactions of the PDHc are:
The reactions of the PDHc serve to interconnect the metabolic pathways of glycolysis, gluconeogenesis and fatty acid oxidation to the TCA cycle. The activity of the PDHc is also important in regulating the flux from glucose to malonyl-CoA which is required for the de novo synthesis of fatty acids. Acetyl-CoA, produced in the mitochondria via the action of the PDHc, will be transported out into the cytosol in the form of citrate when the energy charge of the cell rises. In the cytosol, the citrate is hydrolyzed by ATP-citrate lyase (gene symbol: ACLY) yielding oxaloacetate and acetyl-CoA. In the cytosol the acetyl-CoA can be converted to malonyl-CoA via the action of acetyl-CoA carboxylase (ACC). This malonyl-CoA serves as the precursor for the synthesis of fatty acids. Accumulation of cytosolic malonyl-CoA, as will happen in energy rich conditions, partitions cytosolic fatty acids away from the oxidizing machinery of the mitochondria by inhibiting carnitine palmitoyltransferase I (CPT I). This effect of malonyl-CoA, derived from acetyl-CoA, couples increased rates of glucose oxidation to inhibition of fatty acid oxidation. There is no pathway in mammals for the conversion of acetyl-CoA to glucose and therefore, the activity of the PDHc is highly regulated by a variety of allosteric effectors and by covalent modification. The importance of the PDHc to the maintenance of homeostasis is evident from the fact that although diseases associated with deficiencies of the PDHc have been observed, affected individuals often do not survive to maturity. Since the energy metabolism of highly aerobic tissues such as the brain, skeletal muscle, and the heart is dependent on normal conversion of pyruvate to acetyl-CoA, aerobic tissues are most sensitive to deficiencies in components of the PDHc. Most genetic diseases associated with PDHc deficiency are due to mutations in either of the two subunits (α or β) of the functional PDH heterotetramer. The main pathologic result of such mutations is moderate to severe cerebral lactic acidosis and encephalopathies.
Factors regulating the activity of the PDHc. PDH activity is regulated by its state of phosphorylation, being most active in the dephosphorylated state. Phosphorylation of PDH is catalyzed by four specific PDH kinases, designated PDK1, PDK2, PDK3, and PDK4. The activity of these kinases is enhanced when cellular energy charge is high which is reflected by an increase in the level of ATP, NADH and acetyl-CoA. Conversely, an increase in pyruvate strongly inhibits PDH kinases. Additional negative effectors of PDH kinases are ADP, NAD+ and CoASH, the levels of which increase when energy levels fall. The regulation of PDH phosphatases (PDPs) is less well understood but it is known that Mg2+ and Ca2+ activate the enzymes and that they are targets of insulin action. In adipose tissue, insulin increases PDH activity and in cardiac muscle PDH activity is increased by catecholamines. Green arrows denote activation effects, whereas red T-lines denote inhibitory actions.
The activity of PDH (E1) is rapidly regulated by phosphorylation and dephosphorylation events that are catalyzed by PDH kinases (PDKs) and PDH phosphatases (PDPs), respectively. Phosphorylation of PDH results in inhibition of activity, whereas, dephosphorylation increases it. The PDKs have been shown to be tightly bound the the lipoyl domains of E2, anchoring the kinases to the PDHc. On average, there are two to three PDKs bound to each PDHc.
Phosphorylation of the α subunit of PDH (E1α; encoded by the PDHA1 gene) occurs at three different serine residues termed site 1 (Ser-264), site 2 (Ser-271), and site 3 (Ser-203). Phosphorylation of any of these three serine residues results in the inhibition of the PDHc. However, data show that phosphorylation of site 1 occurs most rapidly and site 3 being the least rapidly phosphorylated. Given these differences in phosphorylation rate, site 1 is of primary importance for the acute regulation of the PDHc activity. Four PDK isozymes have been identified in humans: PDK1, PDK2, PDK3, and PDK4. The PDK1 gene is located on chromosome 2q31.1 and is composed of 24 exons that generate two alternatively spliced mRNAs encoding two distinct protein isoforms. The PDK2 gene is located on chromosome 17q21.33 and is composed of 14 exons that generate four alternatively spliced mRNAs encoding three distinct protein isoforms. The PDK3 gene is located on the X chromosome (Xp22.11) and is composed of 12 exons that generate two alternatively spliced mRNAs encoding two distinct protein isoforms. The PDK4 gene is located on chromosome 7q21.3 and is composed of 11 exons that generate encode a 411 amino acid protein.
Analysis of the patterns of expression of the PDKs shows that PDK2 is the most widely expressed with the highest levels of expression seen in heart, liver, and kidney. PDK1 expression is highest in the heart and skeletal muscle. PDK3 expression predominates in lung and testis. PDK4 expression is high in heart, liver, pancreatic islets, and kidney and each of these tissues are capable of high rates of fatty acid oxidation and also express high levels of the transcription factors PPARα and PPARδ which are known to be critical regulators of the expression of genes encoding lipid metabolizing enzymes.
Utilizing mutant forms of PDH it has been possible to identify which sites are phosphorylated by which PDK. All four PDKs have been shown to be able to phosphorylate sites 1 and 2. Site 3 is only phosphorylated by PDK1. PDK4 has a much higher preference for phosphorylation of site 2 when compared to the activities of PDK1, PDK2, and PDK3 at this site. Data from these types of experiments indicates that the activity of PDK2 is necessary for short-term inhibition of the PDHc via phosphorylation of site 1. On the other hand, PDK1 and PDK4 appear to be more important for allowing multisite phosphorylation which in turn prevents reactivation of the PDHc by PDP.
Regulation of the activity of the various PDKs is manifest with isoform specificity. Pyruvate, generated from glycolysis, exerts differential inhibitory actions on the various PDKs. PDK2 is the most sensitive to inhibition by pyruvate, whereas, PDK4 is relatively insensitive. As the ATP/ADP ratios falls (i.e. increases in ADP levels) ADP will exert an allosteric inhibition on the activity of the PDKs. ADP exerts its allosteric inhibition of PDKs both independently and in synergy with pyruvate inhibition. In contrast to the acute inhibition of PDKs by pyruvate, acute activation occurs via products of the PDHc as well as by fatty acid oxidation, namely acetyl-CoA and NADH. PDK2 is the most sensitive to increases in the level of acetyl-CoA in comparison to the other PDK isoforms. The ratio of NADH to NAD+ also exert isoform-specific allosteric regulation of PDKs. PDK4 is highly responsive (activated) to an increased NADH/NAD+, whereas, PDK2 is much less sensitive to this activation.
Two genetically and biochemically distinct PDP isoforms (PDP1 and PDP2) have been characterized. The PDP1 is a Mg2+-dependent and Ca2+-stimulated protein serine phosphatase of the protein phosphatase 2C (PP2C) superfamily that is found bound to the inner mitochondrial membrane. Both the PDP1 and PDP2 phosphatase are heterodimeric enzymes composed of a catalytic subunit (PDPc) and a regulatory subunit (PDPr). There are two different catalytic subunit genes in humans identified as the PDP1 gene and the PDP2 gene. The PDP1 encoded protein (also known as PPM2C) is a component of the PDP1 phosphatase complex, whereas the PDP2 encoded protein (also known as PPM2C2) is a component of the PDP2 phosphatase complex. The PDP1 gene is located on chromosome 8q22.1 and is composed of 6 exons that generate four alternatively spliced mRNAs encoding two distinct protein isoforms. The PDP2 gene is located on chromosome 16q22.1 and is composed of 3 exons that encode a 529 amino acid protein. The PDP regulatory subunit is encoded by the PDPR gene which is located on chromosome 16q22.1 and is composed of 24 exons that generate four alternatively spliced mRNAs that encode three distinct precursor isoforms.
The PDP1 phosphatase complex is widely distributed and is considered a major regulator of the activity of the PDH complex. The mechanism by which Ca2+ stimulates PDPc activity is by increasing its interaction with the PDH complex while also decreasing the Km for Mg2+ of the catalytic subunits of the PDP1 complex. Calcium ion also increases the association of PDP1 catalytic subunits with the phosphorylated E1α subunit of the PDHc. In contrast, PDPr lowers the sensitivity of catalytic subunits to Mg2+. Both PDP1 components are targeted by insulin, which enhances PDPc activity and decreases PDPr negative control, followed by enhanced PDPc sensitivity to Mg2+ and improved overall PDP1 efficiency. In addition, the level of PDP1 activity is affected by pathology with decreased levels seen in diabetes. The activity of the PDP2 phosphatase is less well understood of the two PDP complexes but has been shown to consist of a catalytic subunit that is insensitive to Ca2+ and is 10-fold less sensitive to Mg2+ than the PDPc of PDP1. However, PDP2 is also considered a target of insulin action and the level of PDP2 activity is decreased in diabetes and during starvation.
Two products of the complex, NADH and acetyl-CoA (both of which are also products of fatty acid oxidation), are negative allosteric effectors on the non-phosphorylated and most active form of PDH. These effectors reduce the affinity of the enzyme for pyruvate, thus limiting the flow of carbon through the PDH complex. In addition, as indicated above, NADH and acetyl-CoA are powerful positive effectors on the activity of PDK2 and PDK4. Since NADH and acetyl-CoA accumulate when the cell energy charge is high, it is not surprising that high ATP levels also up-regulate PDK activity, reinforcing down-regulation of PDH activity in energy-rich cells. Note, however, that pyruvate is a potent negative effector on all PDKs, with the result that when pyruvate levels rise, active PDH will be favored even with high levels of NADH and acetyl-CoA.
Concentrations of pyruvate which maintain PDH in the active form are sufficiently high so that, in energy-rich cells, the allosterically down-regulated, high Km form of PDH is nonetheless capable of converting pyruvate to acetyl-CoA. With large amounts of pyruvate in cells having high energy charge and high NADH, pyruvate carbon will be directed to the two main storage forms of carbon (glycogen via gluconeogenesis and fat production via fatty acid synthesis) where acetyl-CoA is the principal carbon donor.back to the top
The TCA cycle showing enzymes, substrates and products. The GTP generated during the succinate thiokinase (succinyl-CoA synthetase) reaction is equivalent to a mole of ATP by virtue of the presence of nucleoside diphosphokinase. The generation of this GTP is referred to a substrate-level phosphorylation. The 3 moles of NADH and 1 mole of FADH2 generated during each round of the cycle feed into the electron transport chain (ETC) of the oxidative phosphorylation pathway. Each mole of NADH leads to 3 moles of ATP and each mole of FADH2 leads to 2 moles of ATP. Therefore, for each mole of pyruvate which enters the TCA cycle, 12 moles of ATP can be generated.
Citrate synthase (gene symbol: CS) catalyzes the first reaction of the TCA cycle which involves the condensation of the methyl carbon of acetyl-CoA with the keto carbon (C-2) of oxaloacetate (OAA). The CS gene is located on chromosome 12q13.3 and is composed of 12 exons that generate two splice variants. These differentially spliced mRNAs are referred to as CSa and CSb. The CSa mRNA lacks exon 2 and encodes a protein of 466 amino acids. The CSb mRNA contains exon 2 and encodes a protein of 400 amino acids. The major CS mRNA found in hepatocytes is the CSa form.
The citrate synthase reaction is often considered the rate-limiting step of the TCA cycle, however, greater control over the overall rate of the cycle is exerted at the isocitrate dehydrogenase catalyzed reaction. The standard free energy of the citrate synthase reaction, –8.0 kcal/mol, drives it strongly in the forward direction. Since the formation of OAA from its precursor, malate, is thermodynamically unfavorable, the highly exergonic nature of the citrate synthase reaction is of central importance in keeping the entire cycle going in the forward direction, since it drives oxaloacetate formation by mass action principals.
When the cellular energy charge increases, the rate of flux through the TCA cycle will decline leading to a build-up of citrate. Excess citrate is used to transport acetyl-CoA carbons from the mitochondrion to the cytoplasm where they can be used for fatty acid and cholesterol biosynthesis. Citrate is transported across the inner mitochondrial membrane by the tricarboxylate transporter encoded by the SLC25A1 gene. Additionally, the increased levels of citrate in the cytoplasm activate the key regulatory enzyme of fatty acid biosynthesis, acetyl-CoA carboxylase (ACC) while at the same time citrate allosterically inhibits the rate-limiting enzyme of glycolysis, PFK-1.
The isomerization of citrate to isocitrate by aconitase is stereospecific, with the migration of the –OH from the central carbon of citrate (formerly the keto carbon of OAA) being always to the adjacent carbon which is derived from the methylene group (–CH2–) of OAA. The stereospecific nature of the isomerization determines that the CO2 lost, as isocitrate is oxidized to succinyl-CoA, is derived from the oxaloacetate used in citrate synthesis.
Humans contain two distinctly different aconitase enzymes, aconitase 1 and aconitase 2. One is a soluble form found within the cytosol and is involved in the iron-mediated regulation of protein synthesis, specifically the translation of the ferritin and the transferrin receptor mRNA (see the Protein Synthesis page). The soluble aconitase 1 is encoded by the ACO1 gene located on chromosome 9q21.1 which encodes a protein of 889 amino acids. The other enzyme (aconitase 2) is the mitochondrial enzyme involved in the TCA cycle. The mitochondrial aconitase 2 is encoded by the ACO2 gene located on chromosome 22q13.2 which encodes a protein of 780 amino acids.
Mitochondrial aconitase 2 is one of several mitochondrial enzymes known as non-heme-iron proteins. These proteins contain inorganic iron and sulfur, known as iron sulfur centers, in a coordination complex with sulfhydryls of cysteine residues in the protein. There are two prominent classes of non-heme-iron complexes, those containing two equivalents each of inorganic iron and sulfur, Fe2S2, and those containing 4 equivalents of each, Fe4S4. Aconitase 2 is a member of the Fe4S4 class. Its iron sulfur centers are often designated as Fe4S4Cys4, indicating that 4 cysteine sulfur atoms are involved in the complete structure of the complex. In iron sulfur compounds the iron is generally involved in the oxidation-reduction events.
There are three different IDH enzymes in humans identified as IDH1, IDH2, and IDH3 with only the IDH3 isoform being involved in the TCA cycle. Isocitrate is oxidatively decarboxylated to 2-oxoglutarate (α-ketoglutarate) by isocitrate dehydrogenase, IDH. It is generally considered that control of carbon flow through the TCA cycle is regulated at IDH by the powerful negative allosteric effectors NADH and ATP and by the potent positive effectors; isocitrate, ADP and AMP. From the latter it is clear that cell energy charge is a key factor in regulating carbon flow through the TCA cycle.
The IDH1 and IDH2 enzymes are NADP-dependent enzymes. There are three distinct NAD+-dependent IDH enzymes expressed in humans. The NAD+-dependent isoforms are all commonly referred to as IDH3. These IDH isoforms are the primary mitochondrial enzymes responsible for oxidative decarboxylation of isocitrate within the context of the TCA cycle. The isocitrate dehydrogenase reaction is the rate-limiting step, as well as the first NADH-yielding reaction of the TCA cycle. The CO2 produced by the IDH3 reaction is from the original C-1 carbon of the oxaloacetate used in the citrate synthase reaction. Each of the functional IDH3 enzymes is a heterotetramer composed of three different subunits identified as the α, β, and γ subunits. The primary composition of IDH3 is α2β1γ1. The α subunit is encoded by the IDH3A gene located on chromosome 15q25.1–q25.2 and is composed of 12 exons that encode a protein of 366 amino acids. The β subunit is encoded by the IDH3B gene located on chromosome 20p13 and is composed of 14 exons that generate three alternatively spliced mRNAs, each of which encode a distinct protein isoforms. IDH3B isoform a is a 385 amino acid precursor protein, isoform b is a 383 amino acid precursor protein, and isoform d is a 376 amino acid precursor protein. The γ subunit is encoded by the IDH3G gene located on chromosome Xq28 and is composed of 12 exons that generate two alternatively spliced mRNAs encoding proteins of 393 amino acids (isoform a) and 380 amino acids (isoform b).
Both IDH1 and IDH2 enzymes utilize NADP+ as their cofactor and generate NADPH via oxidative decarboxylation of isocitrate outside the context of the TCA cycle. The IDH1 is a homodimeric enzyme encoded by the IDH1 gene located on chromosome 2q33.3 and is composed of 12 exons that generate three alternatively spliced mRNAs, all of which encode the same 414 amino acid protein. IDH1 is found primarily in the cytosol and the peroxisomes. The IDH2 enzyme is also a homodimeric enzyme but is located in the mitochondria. IDH2 is encoded by the IDH2 gene located on chromosome 15q26.1 and is composed of 12 exons that generate three alternatively spliced mRNAs, each of which encode a distinct protein isoform. The primary function of IDH1 and IDH2 is to serve as producers of NADPH to enhance the cellular responses to oxidative stress and the generation of reactive oxygen species, ROS. The principal sources of NADPH are from the glucose-6-phosphate dehydrogenase (G6PD) reaction in the pentose phosphate pathway and via malic enzyme which is involved in the mobilization of mitochondrial acetyl-CoA to the cytosol. The significance of IDH1 and IDH2 produced NADPH can be made clear by pointing out that cells with low level IDH1 and IDH2 expression are more sensitive to oxidative stress than cells with higher levels of expression of these two enzymes.
2-Oxoglutarate (also called α-ketoglutarate) is oxidatively decarboxylated to succinyl-CoA by the 2-oxoglutarate dehydrogenase complex, OGDH. The OGDH complex is commonly referred to as the α-ketoglutarate dehydrogenase (α-KGDH) complex. This reaction generates the second TCA cycle equivalent of CO2 and NADH. As with the PDH complex described above, the reactions of the OGDH complex proceed with a large negative standard free energy change. The OGDH complex is very similar to the PDH complex in the intricacy of its protein makeup (E1, E2, and E3 activities), cofactors (TPP, lipoic acid, CoA, FAD, and NAD+), and its mechanism of action. Although the E1 activity of the OGDH complex is not subject to covalent modification as are the E1 components of the PDHc, allosteric regulation of the OGDH complex is indeed effected. The enzyme complex is also regulated energy charge, the NAD+/NADH ratio, and effector activity of substrates and products.
The three activities of OGDH complex are identified as oxoglutarate dehydrogenase (E1k), dihydrolipoamide S-succinyltransferase (DLST or E2k), and dihydrolipoamide dehydrogenase (DLD or E3). The oxoglutarate dehydrogenase subunits of the OGDH complex are encoded by the OGDH gene located on chromosome 7p14-p13 and is composed of 26 exons that generate three alternatively spliced mRNAs that encode three distinct isoforms (isoform 1: 1023 amino acid precursor; isoform 2: 427 amino acid precursor; isoform 3: 1019 amino acid precursor). The DLST subunits are encoded by the DLST gene located on chromosome 14q24.3 and is composed of 15 exons that generate two alternatively spliced mRNAs encoding isoform 1 (453 amino acid precursor) and isoform 2 (166 amino acid precursor). The DLD subunits of the OGDH complex are the same as those found in the PDH complex, i.e. encoded by the same gene.
Succinyl-CoA and 2-oxoglutarate are also important metabolites outside the TCA cycle. In particular, 2-oxoglutarate represents a key anaplerotic metabolite linking the entry and exit of carbon atoms from the TCA cycle to pathways involved in amino acid metabolism. 2-Oxoglutarate is also important for driving the malate-aspartate shuttle. Succinyl-CoA, along with glycine, contributes all the carbon and nitrogen atoms required for the synthesis of heme and for non-hepatic tissue utilization of ketone bodies.
The malate/aspartate shuttle. This shuttle is the principal mechanism for the movement of reducing equivalents (in the form of NADH; highlighted in the red boxes) from the cytoplasm to the mitochondria. The glycolytic pathway is a primary source of cytoplasmic NADH. Within the mitochodria the electrons of NADH can be coupled to ATP production during the process of oxidative phosphorylation. The electrons are "carried" into the mitochondria in the form of malate. Cytoplasmic malate dehydrogenase (MDH) reduces oxaloacetate (OAA) to malate while oxidizing NADH to NAD+. Malate then enters the mitochondria where the reverse reaction is carried out by mitochondrial MDH. Movement of mitochondrial OAA to the cytoplasm to maintain this cycle requires it be transaminated to aspartate (Asp, D) with the amino group being donated by glutamate (Glu, E). The Asp is then transported out of the mitochondria and enters the cytoplasm. The deamination of glutamate generates 2-oxoglutarate, 2-OG, (α-ketoglutarate) which is transported out of the mitochondria to the cytoplasm. All the participants in the cycle are present in the proper cellular compartment for the shuttle to function due to concentration dependent transport. When the energy level of the cell rises the rate of mitochondrial oxidation of NADH to NAD+ declines and therefore, the shuttle slows. GAPDH is glyceraldehyde-3-phosphate dehydrogenase. AST is aspartate transaminase. SLC25A11 is the malate transporter and SLC25A13 is the aspartate/glutamate transporter.
The conversion of succinyl-CoA to succinate, by succinyl-CoA synthetase (SCS), involves the use of the high-energy thioester of succinyl-CoA to drive synthesis of a high-energy nucleotide phosphate (GTP). This process is referred to as substrate-level phosphorylation. In this process, a high energy enzyme-phosphate intermediate is formed, with the phosphate subsequently being transferred to GDP. Mitochondrial GTP is used in a trans-phosphorylation reaction catalyzed by the mitochondrial enzyme nucleoside diphosphokinase to phosphorylate ADP, producing ATP and regenerating GDP for the continued operation of succinyl-CoA synthetase.
Succinyl-CoA synthetase is a heterodimeric enzyme composed of an α (alpha) and a β (beta) subunit. The α-subunit is invariant in all forms of SCS and is encoded by the SUCLG1 (succinate-CoA ligase) gene. The SUCLG1 gene is located on chromosome 2p11.2 and is composed of 9 exons encoding a protein of 346 amino acids. The β-subunit of SCS determines the nucleotide specificity of the enzyme complex. There are two β-subunit genes encoding the A-beta and G-beta subunits. As indicated by the designations, the A-beta subunit determines specificity for ADP/ATP and the G-beta for GDP/GTP. The A-beta subunit is encoded by the SUCLA2 gene located on chromosome 13q14.2 and is composed of 11 exons encoding a protein of 463 amino acids. The G-beta subunit is encoded by the SUCLG2 gene located on chromosome 3p14.1 and is composed of 14 exons encoding a protein of 440 amino acids.
The multi-enzyme complex called succinate dehydrogenase (SDH) catalyzes the oxidation of succinate to fumarate with the sequential reduction of enzyme-bound FAD to FADH2 as well as the associated non-heme-iron. The SDH complex is the same as complex II of the electron transport chain (ETC) of oxidative phosphorylation. In mammalian cells, the electron acceptor is coenzyme Q (CoQ; written as CoQ10 to depict the major form in human cells), a mobile carrier of reducing equivalents that is restricted by its lipophilic nature to the lipid phase of the inner mitochondrial membrane and by its association with subunits of the SDH complex. The SDH complex is the only enzymatic activity of the TCA cycle that is embedded within the inner mitochondrial membrane, all of the other enzymes reside within the matrix of the mitochondria.
The SDH complex is composed of four different proteins encoded by four different nuclear genes identified as SDHA, SDHB, SDHC, and SDHD. The SDH complex is the only component of the mitochondrial electron transport chain (ETC) that is composed solely of proteins encoded by nuclear genes. In addition to these four proteins, that constitute the functional SDH complex (oxidative phosphorylation complex II), at least two additional proteins are required for proper assembly of the complex. These assembly factors are called SDH complex assembly factor 1 (SDHAF1) and 2 (SDHAF2). The A subunit of SDH is a flavoprotein encoded by the SDHA gene located on chromosome 5p15 which is composed of 15 exons the generate two alternatively spliced mRNAs (isoform 1: 664 amino acids; isoform 2: 616 amino acids). The SDHA encoded protein is the enzymatic activity that carries out the oxidation of succinate to fumarate with the simultaneous reduction of FAD to FADH2. The B subunit of SDH is an iron-sulfur protein encoded by the SDHB gene located on chromosome 1p36.1–p35 which is composed of 8 exons encoding a precursor protein of 280 amino acids. The SDHB encoded protein contains three distinct Fe-S (iron-sulfur) centers that facilitate the transfer of electrons from FADH2 to ubiquinone (coenzyme Q: CoQ). The Fe-S centers of SDHB consist of a 2Fe-2S center, which is near the FAD binding site, a 4Fe-4S center, and a 3Fe-4S center. The C subunit of SDH is an integral membrane protein encoded by the SDHC gene located on chromosome 1q23.3 which is composed of 7 exons encoding a protein of 150 amino acids. The D subunit of SDH is also an integral membrane protein encoded by the SDHD gene located on chromosome 11q235 which is composed of 6 exons encoding a protein of 85 amino acids. The inner mitochondrial membrane embedded SDHC and SDHD encoded proteins (referred to as the membrane domain of the SDH complex) anchor the SDHA and SDHB subunits to the inner mitochondrial membrane. The SDHC and SDHD subunits also facilitate interactions with CoQ and electron exchange from the Fe-S centers of the SDHB subunit. The membrane domain of the SDH complex contains a heme b moiety that is bound at the interface between the SDHC and SDHD subunits. The binding of CoQ to the SDHC and SDHD membrane domain occurs on the matrix side of the inner mitochondrial membrane.
The catalytic activity of the SDH complex is controlled by post-translational modifications that include phosphorylation and acetylation. In addition, the catalytic activity of the SDHA subunit is allosterically inhibited by oxaloacetate. As succinate levels rise the inhibitory oxaloacetate is displaced, thereby activating the SDH complex. Phosphorylation of the SDH complex has been shown to occur at two tyrosine residues via the action of FGR kinase, a SRC family member tyrosine kinase. However, these results were obtained via in vitro experiments and the identity of the kianse(s) that phosphorylate the SDH complex in vivo is still unknown. However, the significance of tyrosine phosphorylation, to the activity of the SDH complex, was demonstrated when it was shown that blocking the activity of the mitochondrial tyrosine phosphatase, PTPMT1, results in hyperphosphorylation and activation of the SDH complex. Reversible lysine acetylation of the SDHA subunit has also been shown to be responsible for the attenuation of succinate oxidation to fumarate. When acetylated the activity of SDHA is reduced primarily due to reduction in substrate entry into the enzyme active site. Multiple Lys (K) residues in the SDHA subunit have been shown to acetylated through the use of mass spec. However, similar to the situation for possible SDH kinases, no definitive SDH acetylase has been characterized. The principal enzyme responsible for the deacetylation of the SDHA subunit has been defined. One of the sirtuin family of deacetylases, SIRT3, has been shown to be the major mitochondrial deacetylase controlling the level of SDHA acetylation.
The fumarate hydratase-catalyzed reaction is specific for the trans form of fumarate. The result is that the hydration of fumarate proceeds stereospecifically with the production of L-malate. The fumarate hydratase gene (symbol: FH) is located on chromosome 1q42.1 and is composed of 11 exons that encode two distinct proteins from a single mRNA. The two forms of fumarate hydratase result from the single FH mRNA as a result of alternative translational start site utilization. One form of the enzyme is localized to the cytosol while the TCA cycle enzyme is localized to the mitochondria. The mitochondrial version of fumarate hydratase (a 510 amino acid protein) is extended at the N-terminus due to the use of an alternative AUG start codon relative to AUG start codon used to produce the cytosolic form of the enzyme. The AUG start codon used for the cytosolic fumarate hydratase makes this version of the enzyme 43 amino acids shorter than the mitochondrial version.
Mitochondrial malate dehydrogenase (MDH) catalyzes the "final" reaction of the TCA cycle. L-malate is the specific substrate for MDH. The forward reaction of the cycle, the oxidation of malate, yields oxaloacetate (OAA). In the forward direction the reaction has a standard free energy of about +7 kcal/mol, indicating the very unfavorable nature of the forward direction. As noted earlier, the citrate synthase reaction that condenses oxaloacetate with acetyl-CoA has a standard free energy of about –8 kcal/mol and is responsible for pulling the MDH reaction in the forward direction. The overall change in standard free energy change is about –1 kcal/mol for the conversion of malate to oxaloacetate and on to succinate.
There are two malate dehydrogenases in humans encoded by two different genes. Both forms of the enzyme are required for the operation of the malate-aspartate shuttle. The cytosolic form of MDH is encoded by the MDH1 gene. The MDH1 gene is located on chromosome 2p13.3 and is composed of 11 exons encoding a protein of 353 amino acids. The TCA cycle MDH is a mitochondrial enzyme encoded by the MDH2 gene. The MDH2 gene is located on chromosome 7cen-q22 and is composed of 10 exons encoding a protein of 296 amino acids.
The overall stoichiometry of the TCA cycle is:
At least three of the genes that encode TCA cycle enzymes, or protein subunits of enzyme complexes, have been shown to manifest mutations in certain types of familial cancer syndromes. Mutations in two of the three isocitrate dehydrogenase (IDH) genes expressed in humans have been identified to harbor specific mutations and these mutations are correlated to the development of certain forms of cancer. The most common forms of cancer in which mutations in the IDH1 and IDH2 genes have been discovered are gliomas in the brain. As indicated in the descriptions of the enzymes and enzyme complexes of the TCA cycle, the succinate dehydrogenase (SDH) complex is composed of four subunits encoded by four distinct genes, SDHA, SDHB, SDHC, and SDHD. Mutations in all four of these genes are associated with several familial cancer syndromes. In addition to the four functional components of the SDH complex, mutations in the SDH assembly factor gene, SDHAF2, are also found in certain forms of cancers. Although mutations in the SDH subunit assembly factor gene, SDHAF1, have not been associated with cancer syndromes, they are associated with mitochondrial deficiency syndromes. The SDH complex gene associated cancers include paragangliomas, pheochromocytomas, gastrointestinal stromal tumors (GIST), and an aggressive form of renal cancer identifed as SDH-RCC (SDH-deficient renal cell carcinoma). Cancer syndromes have also been asociated with mutations in the fumarate hydratase (FH; also called fumarase) gene. The FH gene mutation associated cancers are cutaneous and uterine leiomyomas and an aggressive form of type 2 papillary renal cancer. The renal papillary carcinoma is characterized by a metabolic shift to aerobic glycolysis and glutamine-dependent reductive carboxylation. Details of the TCA cycle gene mutations and their associations to cancer syndromes will be discussed in the order in which the enzymes appear within the context of the TCA cycle. It is important to note that succinate dehydrogenase is also complex II of the oxidative phosphorylation pathway and contributing to metabolic shifts seen in SDH-deficient cancers are deficiencies in mitochondrial energy generation via oxidative phosphorylation.
As indicated earlier, both IDH1 and IDH2 enzymes utilize NADP+ as their cofactor and generate NADPH via oxidative decarboxylation of isocitrate outside the context of the TCA cycle. The primary function of IDH1 and IDH2 is to serve as producers of NADPH to enhance the cellular responses to oxidative stress and the generation of reactive oxygen species, ROS. The principal sources of NADPH are from the glucose-6-phosphate dehydrogenase (G6PD) reaction in the pentose phosphate pathway and via malic enzyme which is involved in the mobilization of mitochondrial acetyl-CoA to the cytosol. The significance of IDH1 and IDH2 produced NADPH can be made clear by pointing out that cells with low level IDH1 and IDH2 expression are more sensitive to oxidative stress than cells with higher levels of expression of these two enzymes.
Clinical significance is associated with the IDH1 and IDH2 genes as there has been a close association made between mutations in these two genes and the development of certain forms of cancer, specifically gliomas of the brain. All of the mutations in IDH1 that are associated with gliomagenesis are missense mutations that alter the normal Arg residue at position 132 to a histidine (R132H). The mutations in IDH2 are also single amino acid missense mutations at the comparable Arg residue at position 172. The characterized mutations in IDH1 and IDH2 that are associated with the early onset of gliomagenesis result in altered activities of the encoded proteins. The altered activity leads to the production of 2-hydroxyglutarate (2-HG) and the loss of NADPH production. The R132H mutation in IDH1 and the similar mutation in IDH2 result in a loss of isocitrate binding while at the same time altering the catalytic activity of the enzyme such that it oxidizes 2-oxoglutarate (α-ketoglutarate), its normal product, to 2-HG without NADPH production. Indeed, in gliomas with IDH1 mutations the levels of 2-HG are elevated relative to normal cells. Increases in 2-HG levels coupled with decreases in 2-oxoglutarate levels results in competitive inhibition of several important 2-oxoglutarate-dependent dioxygenases such as DNA demethylases and histone demethylases that are involved in regulating chromatin structure, and prolyl hydroxylase domain (PDH) containing enzymes such as those that are involved in cellular responses to hypoxia.
Mutations in IDH2 are much less common than mutations in IDH1 and the appearance of the mutations in these two genes are mutally exclusive meaning that a glioma with mutated IDH1 does not also harbor a mutant IDH2 gene and visa versa. Although associated with gliomagenesis, gliomas that contain mutant IDH1 or IDH2 genes are associated with a better prognosis than gliomas with wild-type IDH1 or IDH2. In addition to gliomas, the same IDH1 missense mutation is found in acute myelogenous leukemia, cholangiocarcinoma, cartilaginous tumors, prostate cancer, papillary breast carcinoma, acute lymphoblastic leukemia, angioimmunoblastic T-cell lymphoma, and primary myelofibrosis.
Mutations in the genes encoding the four proteins comprising the SDH complex result in a pathophysiological state defined as pseudo-hypoxia. This pathophysiologic state leads to a subsequent increase in angiogenesis, via activation of the HIF-1 hypoxia response pathway, resulting in the ability of tumors to acquire nutrients encouraging their aberrant growth. Activation of the HIF-1 pathway in SDH-deficient tumors is most likely the result of accumulating levels of succinate which is then transported into the cytosol via the action of the mitochondrial dicarboxylate transporter. Accumulation of cytosolic succinate inhibits the 2-oxoglutarate-dependent dioxygenases [prolyl hydroxylase domain (PDH) enzymes] responsible for the hydroxylation of the α-subunit (HIF1α) of HIF-1. The hydroxylation of HIF1α normally occurs continuously under normoxic conditions which stimulates ubiquitination and proteosomal degradation of HIF1α, thereby restricting the transcription factor function of HIF-1. The loss of HIF1α hydroxylation results stabilization of the protein and consequent activation of the HIF-1 pathway in the absence of hypoxia. Activation of HIF-1 in the absence of hypoxia leads to increased angiogenesis within the SDH-deficient tumor environment promoting the growth of the tumor cells. Because loss of function of any one of the four subunits of the SDH complex results in the progression to cancer, all four genes encoding proteins of the complex (SDHA, SDHB, SDHC, and SDHD) have been classified as tumor suppressors.
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumor of the gastrointestinal tract. Until very recently the vast majority of GISTs were associated with mutations resulting in the activation of the KIT gene encoded protein (75% to 80%). Additional activating mutations, associated with GIST, although not as common as the KIT mutations, are those in the PDGF receptor A (PDGFRA: 5% to 8%) gene. Less than 10 years age a new class of GIST was identified that is due to mutations in several of the genes encoding protein components of the SDH complex. The initially characterized SDH-deficient GISTs were found to be associated with mutations in the SDHB, SDHC, and SDHD genes. More recently SDHA deficient GISTs have been characterized. The SDH-deficient GISTs are wild type for both the KIT and PDGFRA genes. The SDH-deficient tumors occur exclusively in the stomach and commonly metastasize to lymph nodes. These SDH-deficient tumors are also charactreized by having a unique epithelioid morphology often with a plexiform and multinodular growth pattern and many of the tumors also overexpress the insulin-like growth factor 1 receptor (IGFR1) gene. Since their initial characterization, the SDH-deficient GISTs have been shown to account for up to 7.5% of these forms of cancer in adult patients and represent the vast majority these types of tumors that are found in childhood.
Mutations in all four SDH complex genes, as well as the SDHAF2 gene, are associated with additional forms of cancer. These additional forms are defined as the hereditary paraganglioma and pheochromocytoma syndrome (HPGL/PCC). Paragangliomas (PGLs) are rare tumors derived from the paraganglia. The paraganglia are neuroendrocrine tissues that are symmetrically distributed along the paravertebral axis from the base of the skull and the neck to the pelvis. In the adult that major paraganglionic organs are the carotid body and the adrenal medulla. The carotid body represents a small cluster of chemoreceptors and associated supporting cells that runs along the bottom of the bifurcation of the carotid artery. The primary function of the carotid body is to detect changes in the partial pressures of O2 and CO2 within the arterial blood, but is also involved in the sensation of pH and temperature. The adrenal medulla secretes the catecholamines, epinephrine and norepinephrine, in response to stress-mediated stimulation of preganglionic sympathetic neurons. Paragangliomas are primarily found in the head and neck region, particularly in the carotid bodies. Paragangliomas arising from chromaffin cells of the adrenal medulla are defined as pheoochromocytomas and they characteristically hypersecrete catecholamine. The paraganglioma-pheochromocytoma syndrome (HPGL/PCC) was originally identifed as being caused by mutations in the SDHB, SDHC, and SDHD genes and later to be associated with SDHA and SDHAF2 gene mutations.
Similar to the alterations that occur in metabolic programs in SDH-deficient cancers, FH-deficient cells exhibit a substantial metabolic reorganization. FH-deficient tumor cells satisfy their need for high rates of ATP production by developing high rates of glucose uptake and higher rates of glucose oxidation via glycolysis. This form of metabolic reprogramming in cancer cells (as well as that occuring in normal rapidly proliferating cells) is referred to as the Warburg effect. The reduced entry of glycolytic intermediates into the TCA cycle, due to FH deficiency, results in diversion of glucose to lactate as well as into other metabolic pathways of biomass production. All of these shifts in metabolism are not unique to FH-deficient tumors but are a common occurrence in most tumors. As indicated in the Glycolysis page and in the section above on SDH-deficient tumors, the common changes in metabolic profiles in cancer cells is, in part, the result of the activation of the hypoxia induced factor 1 (HIF-1) transcriptional program.
When fumarate accumulates in the mitochondria of FH-deficient cells the metabolite is transported to the cytosol by the dicarboxylate transporter. Within the cytosol the accumulating fumarate, similar to the effect of accumulating succinate, results in the inhibition of 2-oxoglutarate-dependent dioxygenases that are responsible for the hydroxylation of the α-subunit (HIF1α) of the HIF-1 complex. The HIF1α proline hydroxylases are commonly referred to as the prolyl hydroxylase domain (PDH) enzymes. The hydroxylation of HIF1α presents binding sites for the VHL ubiquitin ligase which then ubiquinates HIF1α resulting in its proteosomal degradation. Inhibition of the PDH enzymes by fumarate results in loss of HIF1α turnover which then allows the HIF-1 complex to migrate to the nucleus where a program of genes are activaed that are normally responsible for cell survival in response to hypoxia. One of the major responses to HIF-1 transcriptional activity is increased angiogenesis allowing tumor cells higher rates of access to biomolecules for growth. Additional metabolic changes in FH-deficient tumors have been shown to be the result of reduced activation and reduced expression of the master metabolic regulatory kinase, AMPK. The loss of AMPK activity results in increased rates of lipid and protein synthesis in the tumor cells.
Mutations in the fumarate hydratase (FH: also called fumarase) gene have been shown to be associated with hereditary leiomyomatosis and renal cell carcinoma (HLRCC). Leiomyoma is a form of smooth muscle tumor that can develop in any organ but is most commonly found in the uterus, esophagus, and small intestine. Uterine fibroids are a common form of leiomyoma of the uterine smooth muscle that are benign. HLRCC is an autosomal dominant hereditary cancer syndrome that is characterized by a predisposition to the development of cutaneous and uterine leiomyomas and a very aggressive form of papillary kidney cancer. HLRCC-associated renal tumors readily metastasize to both regional and distant lymph nodes making this form of renal cancer significantly more aggressive than other forms of genetically defined renal cancer. Mutations resulting in loss of function of fumarate hydratase are the primary genetic alterations associated with HLRCC. The FH mutations found in HLRCC are missense, frameshift, partial deletions, or complete deletions. Because loss of function of the FH gene results in the progression to cancer, the gene has been classified as a tumor suppressor.back to the top
There is no direct hormonal regulation of TCA cycle activity, substrate availability and allosteric regulation of enzyme activity controls the rate of flux through the cycle. Fuel enters the TCA cycle primarily as acetyl-CoA, albeit several intermediates in the cycle are replenished via the process referred to as anapleurosis. The generation of acetyl-CoA from carbohydrates is a major control point of the cycle. This is the reaction catalyzed by the PDH complex.
By way of review, the PDH complex is inhibited by acetyl-CoA and NADH and activated by non-acetylated CoA (CoASH) and NAD+. The pyruvate dehydrogenase activities of the PDH complex are regulated by their state of phosphorylation. This modification is carried out by specific kinases (PDK1–PDK4) and the phosphates are removed by a specific phosphatase (PDH phosphatase, PDP). The phosphorylation of PDH inhibits its activity and, therefore, leads to decreased oxidation of pyruvate. PDKs are activated by NADH and acetyl-CoA and inhibited by pyruvate, ADP, CoASH, Ca2+ and Mg2+. PDP, in contrast, is activated by Mg2+ and Ca2+.
Since three reactions of the TCA cycle as well as PDH utilize NAD+ as co-factor it is not difficult to understand why the cellular ratio of NAD+/NADH has a major impact on the flux of carbon through the TCA cycle. Substrate availability can also regulate TCA flux. This occurs at the citrate synthase reaction as a result of reduced availability of oxaloacetate. Product inhibition and energy state also controls the TCA flux. The major control point in the TCA cycle occurs at the IDH3 catalyzed reaction. Both NADH and ATP allosterically inhibit the enzyme. The IDH3 catalyzed reaction is, therefore, considered the rate-limiting reaction of the cycle. Other points of control include citrate inhibition of citrate synthase and NADH and ATP allosteric inhibition of 2-oxoglutarate dehydrogenase (α-ketoglutarate dehydrogenase). Several key enzymes of the TCA cycle are also regulated allosterically by Ca2+, ATP, and ADP.back to the top