Metabolism. TCA, Citric Acid

Metabolism Part 11. The TricarЬoxylic Acid (Т СА), Citric Acid, or Krebs Cycle In 1935, Albert Szent-Gyorgyi found that four-carbon di­ carboxylate ions such as the succinate, fumarate, or malate ions catalyzed the uptake of 02 when added to suspensions of minced pigeon-breast muscle tissue and postulated that these intermediates were linked Ьу а sequence of enzyme­ catalyzed reactions, succinate - fumarate - malate - oxaloacetate Shortly thereafter, Carl Martius and Franz Knoop extended this pathway Ьу outlining the steps Ьу which these suspen­ sions oxidize the citrate ion to succinate, citrate - isocitrate - a-ketoglutarate - succinate In 1937, Hans Krebs found that citrate is readily formed from oxaloacetate and suggested that this pathway was in fact а cycle of reactions that was responsiЫe for the aeroЬic oxidation of fuel molecules. Concern over whether citric acid (or more accurately the citrate ion) was the first product of this cycle led Krebs to propose calling this sequence of en­ zyme-catalyzed reactions the tricarboxylic acid cycle. The tricarboxylic acid (ТСА), citric acid, or Krebs cycle differs from glycolysis in several ways. First, and foremost, it is а cyclic rather than linear pathway that catalyzes the oxidation of fuel molecules without а net production or con­ sumption of the intermediates along the pathway. Second, glycolysis occurs in the soluЫe portion of the cell cytoplasm, whereas the enzymes that catalyze the ТСА cycle are bound to the inner walls of the mitochondria. Third, anaeroЬic glycolysis can recycle its NAD+ coenzymes and therefore stand on its own. The ТСА cycle cannot stand alone; it is tightly coupled to electron transport and oxidative phos­ phorylation whose enzymes are also found on the inner walls of the mitochondria. This reaction is too complex for а single enzyme and is catalyzed Ьу а pyruvate dehydrogenase complex which con­ tains three enzymes and five coenzymes. Two of the enzymes catalyze oxidation-reduction reactions (pyruvate dehydro­ genase and dihydrolipoyl dehydrogenase), and the third cat­ alyzes the transfer of an acetyl group (dihydrolipoyl transa­ cetylase). The five coenzymes are built from essential vita­ mins or "vital amines". Three of the coenzymes act as oxidizing/reducing agents (lipoic acid, flavin adenine dinu­ cleotide or FAD, and nicotinamide adenine dinucleotide or NAD+ ). А fourth coenzyme activates pyruvate for the loss of СО 2 (thiamine pyrophosphate or ТРР), and the fifth acts as an acetyl group carrier (coenzyme А). The reactions carried out Ьу the pyruvate dehydrogenase complex are shown in Figure 1. The first step involves attack on the pyruvate ion Ьу а thiamine pyrophosphate (ТРР) coenzyme bound to the pyruvate dehydrogenase enzyme. The С-Н group in the thiazole ring at the heart of the ТРР coenzyme is acidic enough to add across the carbonyl group of the pyruvate ion. This intermediate then loses СО 2 to form а carbanion that is staЬilized Ьу the positively charged nitrogen on the thiazole ring. Тhе Brldge Ьetween Glycolysls ancl the ТСА Cycle The end product of glycolysis under aeroЬic conditions is the pyruvate ion. The starting material for the ТСА cycle, however, is а substance known as acetyl coenzyme А. The bridge between these pathways therefore involves convert­ ing pyruvate into acetyl СоА, о 11 о 11 СН3ССO2- - СНзС-8-СоА FADH Formally, at least three things occur in this reaction: decar­ boxylation of an a-ketocarboxylic acid to an aldehyde, о 11 о FAD 11 СН3ССО2Н - СНзСН + СО2 oxidation of the aldehyde to а carboxylic acid, X NAD + NAD Н,Н о снJн - СНзСО2Н and reaction between the carboxylic acid and а thiol to form а thioester, СНзСО2Н + HS-CoA - СНзСО-8-СоА (Acotyl СоА) {Dihydrolipoic Acid} Figure 1. The reac1ions catalyzed Ьу the pyruvate dehydrogenase complex that bridge the gap Ьetween glycolysis and the tricarЬoxyllc acid cycle. The carbanion is now oxidized Ьу а lipoic acid coenzyme Ьound through the carЬonyl group to the dihydrolipoyl trans­ acetylase enzyme. The net effect of this reaction is simulta­ neously to regenerate the ТРР coenzyme, reduce lipoic acid to dihydrolipoic acid, oxidize the carbanion to an acetyl group, and Ьind the acetyl group to the reduced form of lipoic acid as а thioester. The transacetylase enzyme then catalyzes the transfer of the acetyl group from dihydrolipoic acid to coenzyme А. Once again, the acetyl group is carried as а thioester, and acetyl СоА is therefore written as СНзСО-8-СоА. Dihydrolipoic acid is oxidized back to lipoic acid Ьу an FAD coenzyme bound to the dihydrolipoyl dehydrogenase enzyme, and the reduced FADH2 coenzyme is then oxidized back to FAD Ьу NAD +. This sequence of five reactions therefore transforms pyruvate to acetyl СоА with the net loss of а molecule of СО2 and the net reduction of one NAD+ coenzyme to NADH. Why SJnlhe8ln AcelJI C08nzJme А? Why does the cell convert pyruvate into acetyl СоА? It would Ье much easier to build an enzyme that simply cata­ lyzed the oxidative decarЬoxylation of pyruvate to the ace­ tate ion, СНзСОСО2 - - CH3C02 - acetyl СоА. Because the pathways for the oxidation of carЬo­ hydrates, proteins, and lipids all converge at the first step of the ТСА cycle where acetyl СоА is consumed, the ТСА cycle is often called the central pathway of aeroblc metabolism. Тhе TrlcarЬoxyllc Acld Cycle Step 1: Aldol Condвnsвtion The first step in the ТСА cycle shown in Figure 2 is an aldol condensation of acetyl СоА and oxaloacetate to give an enzyme-bound thioester intermediate. Hydrolysis of this thioester gives the citrate ion and coenzyme А. acetyl СоА о 11 СоА-8-С-СНа СО2 1� _ __. C02 o:uloacetate СО 2 6Н2 НО-6-со2 - + HS-CoA The advantage of making acetyl СоА revolves around the fact that thioesters have large enough standard free energies of hydrolysis to Ье included in the class of high-energy com­ pounds. Ву coupling the synthesis of а thioester with the oxidative decarboxylation reaction, some of the energy re­ leased in this reaction can Ье captured. Olher Sources of Ac:etyl C08nzJme А The degradation of carЬohydrates such as glucose is not the only source of acetyl СоА. Catabolism of the amino acids lysine, leucine, isoleucine, tryptophan, phenylalanine, and tryosine also forms acetyl СоА. Furthermore, the ,8-oxida­ tion spiral used to break down fatty acids such as steric acid, СНз(СН2)16СО2Н, involves oxidation of these fatty acids to о •сн_�-S-СоА Acetyl СоА Щ �-sн. 2Н c;x>i C=oi..J. �Н. н,о но-с\-с ?°" J-Сн2 ¼ 1 Н.О�Е ,,..fOi- CoA-SH,GТP GOP • F'<>; 1!14 щ lиcitrate �u •t�i '""сн,, Succinate �� '""t [E�Oz! at. Оаоас� c;x>i МOlate с\-н н-� Fumarate 2Н�со; "?О. •� a-Kela9lutarate "СО, [ С =аО $-Сад Succinyt СоА Сад-SН со2 ,2н �о; · "fO, "fН. H!:� � у-н.о -� -�о; н-с-со; Oz Figure 2. The tricarЬoxylic acid cycle. The asterisks mark labeled acetyl Сод injected into this cycle. Oxalaeuccinate the fate of 1 co2citrate да 0 = -32 kJ/mol ' This reaction is catalyzed Ьу an enzyme known as condens­ ing enzyme or citrate synthetase, and the equilibrium lies heavily on the side of the products (Кс = 4.4 Х 105) Ьecause the free energy of hydrolysis of the thioester drives the reaction to completion. Step 2: Cltrate lsomвrlzstlon The symmetric citrate ion is now converted into an opti­ cally active isomer. Тhiв reaction is catalyzed Ьу an enzyme known as aconitase Ьecause it is presumed to равв through an enzyme-bound intermediate known as the cis-aconitate ion. According to this hypothesis, aconitase catalyzes а re­ versiЫe dehydration/hydration reaction which moves the -ОН group from the third to the fourth carbon atom of the citrate ion. ?н н,о н,о -o�-CH:i-C-CH:i-C02 ,,! [-02C-CH:i-C=CH-C02 -J � 1 1 СО 2 co2 cis-aconitate citrate ?Н -02C-CH:i-CH-CH-C02 C02 isocitrate да о • = 6 kJ/mol НО-С-н � � 2Н Ьо, со, cla- 6н2 1 "C­ The net effect of this reaction is to convert а 3 ° alcohol, which cannot undergo oxidation, into а 2 ° alcohol that сап Ье oxidized. Stвp З: Oxidation of lsocltratв The isocitrate ion is now oxidized Ьу NAD + in а reaction catalyzed Ьу isocitrate dehydrogenase to form an enzyme­ bound intermediate which loses СО 2 to give the a-ketoglu­ tarate ion. н NAD+ NADH • Н+ -02C-CH,,-CH-CH-C02-U ОН он 1 -02С-С=С-СО2- + Н2О - -02с-сн.-tн-со21 1 1 C02isocitrate н о о со, 11 11 [-О2С-СН2-СН-С-СО2-\}. -02С-СН,,-СН,,-С-СО2a-ketoglutarate �О2_ oxalosuccinate дG 0 ' = 21 kJ/mol The loss of С02 сап Ье understood Ьу noting that the pres­ ence of а carboxylate ion on С3 and а carЬonyl on С2 makes the intermediate а P-ketoacid, and P-ketoacids readily un­ dergo decarboxylation reactions. Stвp 4: Oxldation of a-Квtoglutaratв The a-ketoglutarate ion is now transformed to succinyl СоА Ьу а series of reactions which proceed Ьу the same five­ step mechanism as the degradation of pyruvate to acetyl СоА. о 11 -О2С-СН,,-СН,,-С- СО2- + NAD+ + HS-CoA a-ketoglutarate о 11 malate дG 0 = 4 kJ/mol ' Stвp В: Oxldatlon of Мв/аtв Malate dehydrogenase then catalyzes the NAD + oxidation of L-malate to give а dicarЬoxylate ion which сап Ье thought of as the product of а fusion of the oxalate and acetate ions, thus the name oxaloacetate. он -02С-СН2-СН-СО2- + NAD + malate о -02С-СН2-�-СО2- + NADH + н + oxaloacetate дG 0 = +30 kJ/mol ' This energetically unfavoraЫe reaction is pulled to comple­ tion Ьу the rapid rate at which oxaloactate condenses with acetyl СоА to form citrate, thereby reactivating the cycle. 1he Rнull8 of l8oloplc LaЬ811ng Experlments -o2C-CH,,-CH,,-C-S-CoA + СО2 + NADH + н+ succinyl СоА дG 0 ' = 33 kJ/mol Once again, the cell captures some of the energy given off in an oxidative decarboxylation reaction to synthesize а high­ energy thioester bond. Stвp 5: Нydrolysls of Succlnyl СоА The standard free energy of hydrolysis of thioesters is large enough to drive substrate-level phosphorylation in which АТР is created from ADP and the phosphate ion. The first step in this process involves capturing the energy given off during hydrolysis of the thioester to form GТР (guano­ sine triphosphate), о н,о 11 CoA-S-C-CH,,-CH,,-C02- + GDP3- + НРО42- J... succinyl СоА Asterisks are used in Figure 2 to follow the fate of isotopi­ cally laЬeled acetyl СоА through the ТСА cycle. lf both carbon atoms of the acetyl group in acetyl СоА were initially labeled with 14С, the citrate ion formed in the first reaction would Ье labeled at С1 and С2 • Because the citrate ion is symmetric, it seems just as likely that aconitase would move the -ОН group on С3 to the isotopically labeled С2 and unlabeled С4 positions. Experimentally, we find that acon­ itase invariaЬly transfers the -ОН group to the unlabeled С4 position. This raises an interesting question: How does aconitase catalyze an asymmetric reaction on the symmetric citrate ion? The citrate ion is symmetric or achiral because the two ends of the molecule are mirror images of each other. The top and bottom halves of the molecule are different, howev­ er, and the citrate ion is therefore prochiral; it has the poten­ tial for acting as if it were chiral. си.со.и -02С-СН,,-СН2-СО2- + GTP•- + CoA-SH succinate дG 0 = -3 kJ/mol which сап then Ье used to convert ADP to АТР. GTP4- + ADP3- - GDP3- + AТP4' Step 8: Oxldatlon of Succ/natв The last three steps are designed to recover oxaloacetate. Succinate is first oxidized to fumarate Ьу an FAD coenzyme covalently bound to the su�cinate dehydrogenase enzyme. -02С-СН2-СН2-СО2- + FAD succinate fumarate н си.со.и top bottom Asymmetric enzymes such as aconitase сап recognize and preferentially Ьind to one end of this molecule and thereby catalyze asymmetric reactions on what appears to Ье а sym­ metric substrate. 1he Net Effect of the ТСА Cycle А single turn around the ТСА cycle produces one molecule of АТР, two molecules of СО 2, and four reduced NADH or FADH2 coenzymes. СНзСО--S-СоА + 3 NAD + + FAD + ADP3- + НРО4 - + 2 Н20 -2 СО2 + HS-CoA + 3 NADH + FADH2 + АТР•- + 2 Н + 2 -02c-t4-co2- + FADH2 н fumarate дG 0 ' "' О kJ/mol Stвp 7: Нydratlon of Fumaratв Fumarate hydratase or fumarase then catalyzes the ste­ reospecific addition of water to form the L-malate ion. The NADH and FADH2 coenzymes are oxidized back to NAD + and FAD in а complex series of reactions known as electron transport that eventually reduce 02 to water. NADH + н + + ½02 -NAD + + Н20 FADH2 + ½02 -FAD + Н 0 2 Electron transport is tightly coupled to а process known as oxidative phosphorylation which captures, in the form of АТР, some of the energy given off during this oxidation. Three АТР are produced each time an NADH coenzyme is recycled, and two АТР result from the oxidation of an FADH2coenzyme. When the АТР produced Ьу recycling the NADH and FADH2 coenzymes is added to the АТР synthesized during the hydrolysis of succinyl СоА, the net effect of а single tum around the ТСА cycle is 12 molecules of АТР. 3(NADH - NAD+) 3 х 3 = 9 АТР l(FADH2 - FAD) 1 х 2 = 2 АТР 1 (succinyl СоА - succinate) 1 Х 1 = 1 АТР total = 12 A'rP/CH3CO--S-COA А fourth NAD coenzyme was reduced to NADH when py­ ruvate was oxidized to acetyl СоА in the step bridging glycol­ ysis and the ТСА cycle.Three more АТР are produced when this coenzyme is recycled, and а total of 15 АТР are there­ fore synthesized for each pyruvate ion oxidized in the mito­ chondria of the cell. Another АТР was produced when phosphoenolpyruvate was hydrolyzed to the pyruvate ion during glycosis, and an NADH coenzyme was produced when glyceraldehyde 3phosphate was oxidized to 1,3-diphosphoglyceric acid. Be­ cause glycolysis occurs in the cytosol but oxidation of the NADH coenzyme Ьу electron transport occurs within the mitochondria, recycling of the NADH coenzyme from gly­ colysis is slightly less efficient.0nly two АТР are produced when this coenzyme is oxidized to NAD + . А total of 18 АТР therefore result from the degradation of а single glyceraldehyde 3-phosphate ion. Because each glu­ cose molecule gives two of these ions, oxidation of glucose Ьу glycolysis coupled with the ТСА cycle generates а total of 36 molecules of АТР.Complete oxidation of glucose to СО2 and Н2О therefore has the potential for liberating 18 timeв as much energy as the anaeroblc degradation of glucose to lactate or ethanol described in the first paper in thiв series. is also somewhat misleading Ьecause it leadв one to believe that some of the 02 conвumed in this reaction iв incorporat­ ed into СО2, whereas in practice all of the 02 is reduced to water. А total of 12 molecules of Н20 are actually produced for each molecule of glucose conвumed in thiв reaction. Each glucose molecule iв вplit into two glyceraldehyde 3-phos­ phate ions; there are вiх places where either NAD + or FAD coenzymes are reduced each time а glyceraldehyde 3-phos­ phate ion is oxidized; and а molecule of water iв generated each time one of these coenzymes is recycled. NADH + н + + ½0 2 - NAD + + Н20 FADH2 + ½0 2 -FAD + Н20 + Enlclency о1 Аеrоыс Oxldatlon о1 Glucoee The standard free energy for the oxidation of glucose to C02andH20, дао = -2,870 kJ/mol iв often compared to the вtandard free energy of hydrolysiв ofATP дао = -31 kJ/mol to estimate the efficiency with which electron transport and oxidative phosphorylation capture energy.Assuming that 36 АТР are produced per molecule of glucose consumed, these standard free energies suggest that slightly less than 40% of the energy given off is captured as АТР. This calculation is miвleading, however, Ьесаuве the con­ centrations of metabolic intermediates in Ьiological systems are far from the вtandard state concentration of 1.00 ...М. When one adjusts the free energy of hydrolysis of АТР to reflect more accurately concentrations of АТР, AD P, and phosphate in а typical celP, 36 АТР coпespond to the cap­ ture of as much as 65% of the energy released during the oxidation of glucose. 2 Тhе Balancecl Chemlcal Equatlon for the Oxldatlon о1 Glucose The balanced chemical equation for the oxidation of glu­ cose, The overall stoichiometry of the reaction сап Ье underвtood Ьу noting that water is аlво conвumed in the ТСА cycle.0ne molecule of water iв conвumed during the hydrolysis of the thioester intennediate produced Ьу the aldol condensation in Step 1. А вecond water molecule iв conвumed during the · hydrolysis of succinyl СоА in Step 5, and а third molecule of water is consumed during the hydration of fumarate in Step 7.А total of six water molecules are therefore consumed for each molecule of glucose that enters the ТСА cycle. The roles of oxygen and water in the aeroblc degradation of glucose сап best Ье appreciated Ьу following the fate of isotopically labeled 02 . СвН,2Ов + 6 02* + 6 Н2О -6 СО2 + 12 Н20* Water provides the oxygen atoms necessary to oxidize glu­ cose to СО2, and all of the molecular oxygen is reduced to water. Anaplerotlc Reactlons There are several points on the ТСА cycle where key intermediates are removed to provide the building Ыocks for the Ьiosynthesis of carbohydrateв, proteins, lipidв, nucleic acids, and specialized moleculeв such as chlorophyll.0xalo­ acetate and a-ketoglutarate, for example, are starting mate­ rials for the synthesis of amino acids such as arginine, aspar­ agine, aspartic acid, glutamic acid, glutamine, isoleucine, lysine, methionine, proline, and threonine. Citrate iв used for the Ьiosyntheвiв of both fatty acidв and heme proteinв, and вuccinyl СоА is the starting material for the syntheвis of the pyrrole rings found in hemoglobln, chlorophyll, and the cytochromes. N ot вurprisingly, there are аlво points where key interme­ diates are injected into the ТСА cycle when Ьiomolecules are degraded. 0xaloacetate, a-ketoglutarate, succinyl СоА, and fumarate, for example, are all products of the break down of amino acids. Because it effectively acts as а catalyst to oxidize acetyl СоА to СО2 and Н2О, the ТСА cycle cannot, Ьу itself, pro­ duce or consume any of these intermediateв. The only way to either repleniвh pools of depleted ТСА intermediateв or metaЬolize intermediateв present in excess is through ana­ plerotic (literally: filling up) reactions which provide short­ cuts across the cycle. The most important example of anaplerosis is а reversiЫe reaction in which pyruvate is converted to oxaloacetate Ьу the enzyme pyruvate carboxylase. о 11 СНз-С-СО2- + СО2 +АТР4 - + Н20 pyruvate о -02С-СН2-�-СО2- + ADP 3 - + НРо. 2- + 2 н + oxaloacetate да с , = 2 kJ/mol This reaction compensates for the drain оп the ТСА cycle