Metabolism: Citric Acid Cycle & Related Pathways Citric acid cycle a subset of the reactions of the cycle to pro- The primary catabolic pathway in the body duce a desired molecule rather than to run the is the citric acid cycle because it is here that entire cycle (see HERE). oxidation to carbon dioxide occurs for breakdown products of the cell’s major build- Acetyl-CoA ing blocks - sugars, fatty acids, and amino The molecule “feeding” the citric acid cycle is acids. The pathway is cyclic (Figure acetyl-CoA and it can be obtained from py- 6.63) and thus, doesn’t really have ruvate (from glycolysis), from a starting or ending point. All of YouTube Lectures fatty acid β-oxidation, from ke- the reactions occur in mitochon- by Kevin tone bodies, and from amino HERE & HERE dria, though one enzyme is em- acid metabolism. Molecules bedded in the organelle’s inner from other pathways feeding into membrane. As needs change, cells may use the citric acid cycle for catabolism make the 541 citric acid cycle ‘cataplerotic’. It is worth cle intermediates are also important in amino noting that acetyl-CoA has very different acid metabolism (Figure 6.63), heme syn- fates, depending on the cell’s energy status/ thesis, electron shuttling, and shuttling of needs (see HERE). The description below de- acetyl-CoA across the mitochondrial inner scribes oxidation (catabolism) in citric acid membrane. The ability of the citric acid cy- cycle. cle to supply intermediates to pathways gives rise to the term ‘anaplerotic.’ It means ‘to Anabolically, acetyl-CoA is also very impor- fill up.’ Before discussing the citric acid cycle, tant for providing building blocks for synthe- it is important to first describe one important sis of fatty acids, ketone bodies, amino enzyme complex that is a major source of acids and cholesterol. Other citric acid cy- acetyl-CoA for the cycle. Figure 6.63 - Amino acid metabolism and the citric acid cycle. Amino acids boxed in yellow are made from the indicated intermediate. Amino acids in blue are made into the intermediate in catabolism. Image by Aleia Kim 542 Pyruvate decarboxylation The pyruvate dehydrogenase enzyme is a complex of multiple copies of three subunits that catalyze the decarboxylation of pyru- vate to form acetyl-CoA. The reaction mechanism requires use of five coenzymes. Pyruvate dehydrogenase is an enormous com- plex in mammals with a size five times greater than ribosomes. Subunits The three subunits are designated by E1, E2, and E3. E2 is also referred to as dihydroli- Figure 6.64 - E1 Subunit of Pyruvate Dehydrogenase poamide acetyltransferase and E3 is more Wikipedia precisely called dihydrolipoyl dehydroge- Figure 6.65 - Mechanism of action of pyruvate decarboxylation and oxidation by pyruvate dehydrogenase. 543 nase. Confusion arises with the name for E1. tacks the electrophilic ketone carbon on the Some call it pyruvate dehydrogenase and oth- pyruvate, releasing carbon dioxide and creat- ers give it the name pyruvate decarboxy- ing an enol that loses a proton on the carbon lase. We will use pyruvate decarboxylase to become a 1,3 dipole that includes the posi- solely to refer to E1 and pyruvate dehydroge- tively charged nitrogen of the thiamine. The nase only to refer to the complex of E1, E2, and reaction (step A in Figure 6.65) is a non- E3. oxidative decarboxylation. Oxidation of the two carbon hydroxyethyl unit occurs in the The catalytic ac- transfer to the li- tions of pyruvate poamide. dehydrogenase can be broken Reductive down into three acetylation steps, each tak- Reductive acetyla- ing place on one tion occurs next of the subunits. (Step B) as the 2- The steps, se- carbon hy- quentially occur- droxyethyl unit ring on E1, E2, is transferred to and E3, are 1) de- lipoamide on carboxylation E2. (Lipoamide is of pyruvate; 2) the name for a oxidation of the molecule of lip- decarboxylated Figure 6.66 - Oxidized and reduced structures of oic acid cova- product; and 3) lipoamide (lipoic acid linked to lysine) lently attached to transfer of elec- a lysine side trons to ulti- chain in the E2 subunit). In prokaryotes in mately form NADH (Figure 6.65). the absence of oxygen, the hydroxyethyl group is not passed to lipoamide, but instead is re- Catalysis leased as free acetaldehyde , which can ac- The catalytic process begins after binding of cept electrons from NADH (catalyzed by al- the pyruvate substrate with activation of the cohol dehydrogenase) and become etha- thiamine pyrophosphate coenzyme nol in the process of fermentation. In the through formation of an ylide intermediate. presence of oxygen in almost all aerobic organ- The nucleophilic carbanion of the ylide at- 544 isms, the process continues with transfer of ess, electrons from FADH2 are transferred + the hydroxyethyl unit to E2 and con- to external NAD , forming NADH tinuation of the cycle below. YouTube Lectures (Step E) and completing the over- by Kevin all cycle. Then enzyme can then Oxidation step HERE & HERE begin another catalytic round by Transfer of the hydroxyethyl binding to a pyruvate. group from E1 to the lipoamide coenzyme in E2 is an oxidation, with transfer of electrons Pyruvate dehydrogenase regulation from the hydroxyethyl group to lipoamide’s Pyruvate deyhdrogenase is regulated both disulfide (reducing it) and formation on the allosterically and by covalent modifica- lipoamide of an acetyl-thioester (oxidizing tion - phosphorylation / dephosphoryla- it). The acetyl group is then transferred from li- poamide to coenzyme A in E2 (Step C in Figure 6.65), forming acetyl- CoA, which is released and leaving reduced sulf- hydryls on the li- poamide. In order for the enzyme to return to its original state, the disul- fide bond on lipoamide must be re-formed. This occurs with transfer of elec- trons from reduced li- poamide to an FAD cova- lently bound to E3 (Step D). This reduces FAD to FADH2. Figure 6.67 - Regulation scheme for pyruvate dehydroge- Formation of NADH nase (PD) Image by Aleia Kim In the last step in the proc- 545 tion by pyruvate dehydroge- nase phosphatase (PDP). PDK puts phosphate on any one of three serine residues on the E1 subunit, which causes pyruvate ki- nase to not be able to perform its first step of catalysis - the decar- boxylation of pyruvate. PDP can remove those phosphates. PDK is allosterically activated in the mi- tochondrial matrix when NADH and acetyl-CoA concentra- tions rise. Figure 6.68 - Pyruvate dehydrogenase complex with three phosphorylation sites in red marked by Product inhibition arrows. Wikipedia Thus, the products of the pyruvate dehydrogenase reaction inhibit tion. Regulation of pyruvate dehydrogenase, the production of more products by favoring whether by allosteric or covalent mechanisms its phosphorylation by PDK. Pyruvate, a has the same strategy. Indicators of high en- substrate of pyruvate dehydrogenase, inhibits ergy shut down the enzyme, whereas indica- PDK, so increasing concentrations of sub- tors of low energy stimulate it. For allosteric strate activate pyruvate dehydrogenase by re- regulation, the high energy indicators affect- ducing its phosphorylation by PDK. As con- ing the enzyme are ATP, acetyl-CoA, centrations of NADH and acetyl-CoA fall, NADH, and fatty acids, which inhibit it. PDP associates with pyruvate kinase and re- AMP, Coenzyme A, NAD+, and calcium, on moves the phosphate on the serine on the E1 the other hand, stimulate it (Figure 6.67). subunit. Covalent modification Low concentrations of NADH and acetyl-CoA Covalent modification regulation of pyru- are necessary for PDP to remain on the en- vate dehydrogenase is a bit more compli- zyme. When those concentrations rise, PDP cated. It occurs as a result of phosphoryla- dissociates and PDK gains access to the serine tion by pyruvate dehydrogenase kinase for phosphorylation. Insulin and calcium (PDK - Figure 6.67) or dephosphoryla- can also activate the PDP. This is very impor- 546 Figure 6.69 - The citric acid cycle image by Aleia Kim 547 tant in muscle tissue, since calcium is a signal Acetyl-CoA + Oxaloacetate for muscular contraction, which requires en- ergy. Citrate + CoA-SH Insulin also also activates pyruvate kinase and the glycolysis pathway to use internal- In the next reaction, citrate is isomerized to ized glucose. It should be noted that the isocitrate by action of the enzyme called aco- cAMP phosphorylation cascade from the β- nitase. adrenergic receptor has no effect on pyru- vate kinase, though the insulin cascade Citrate does, in fact, affect PDP and pyruvate kinase. Citric acid cycle reactions Isocitrate Focusing on the pathway itself (Figure Isocitrate is a branch point in plants and bacte- 6.69), the usual point to start discussion is ad- ria for the glyoxylate cycle (see HERE). dition of acetyl-CoA to oxaloacetate (OAA) Oxidative decarboxylation of isocitrate by to form citrate. Acetyl-CoA for the pathway can come from a vari- ety of sources. The reaction join- ing it to OAA is catalyzed by cit- rate synthase and the ∆G°’ is fairly negative. This, in turn, helps to “pull” the malate de- hydrogenase reaction preced- ing it in the cy- Figure 6.70 - Succinyl-CoA synthetase mechanism cle. 548 isocitrate dehydrogenase produces the catalyzed by α-ketoglutarate dehydroge- first NADH and yields α-ketoglutarate. nase. + Isocitrate + NAD The enzyme α-ketoglutarate dehydrogenase is structurally very similar to pyruvate dehy- drogenase and employs the same five coen- α-ketoglutarate + NADH + CO2 zymes – NAD+, FAD, CoA-SH, thiamine This five carbon intermediate is a branch pyrophosphate, and lipoamide. point for synthesis of the amino acid gluta- mate. In addition, glutamate can also be Regeneration of oxaloacetate The remainder of the citric acid cycle involves made easily into this intermediate in the re- conversion of the four carbon succinyl-CoA verse reaction.