TCA Cycle Accounts for More Than 2/3 of ATP Generated from Fuel Oxidation

BC Chap 20 Tricarboxylic Acid Cycle  TCA cycle accounts for more than 2/3 of ATP generated from fuel oxidation  Pathways for oxidation of fatty acids, glucose, amino acids, acetate, and ketone bodies all generate acetyl-CoA, which is substrate to start TCA cycle

o Activated 2-carbon acetyl group oxidized to 2 molecules CO2, and energy consderved as NADH, FAD(2H), and GTP (guanosine triphosphate)

o NADH and FAD(2H) subsequently donate electrons to O2 via electron-transport chain, with generation of ATP from oxidative phosphorylation  TCA cycle central to energy generation from cellular respiration  In TCA cycle, oxidative decarboxylation of α-ketoglutarate catalyzed by multisubunit α-ketoglutarate dehydrogenase complex, which contains coenzymes thiamine pyrophosphate (TPP), lipoate, and flavin adanine dinucleotide (FAD)  Pyruvate dehydrogenase complex (PDC) catalyzes oxidation of pyruvate to acetyl-CoA, thereby providing link between pathways of glycolysis and TCA cycle  2-carbon acetyl group is ultimate source of electrons that are transferred to nicotinamide adenine dinucleotide + (NAD ) and FAD and also the carbon in the 2 CO2 molecules that are produced  Oxaloacetate used and regenerated in each turn of cycle; however, when cells use intermediates of TCA cycle for biosynthetic reactions, carbons of oxaloacetate must be replaced by anaplerotic (filling-up) reactions, such as pyruvate carboxylase reaction  TCA cycle occurs in mitochondrion, where flux tightly coordinated with rate of electron-transport chain and oxidative phosphorylation through feedback regulation that reflects demand for ATP o Rate of TCA cycle increased when ATP utilization in cell increased through response of several enzymes to ADP levels, NADH/NAD+ ratio, rate of FAD(2H) oxidation, or Ca2+ concentration . For example, isocitrate dehydrogenase allosterically activated by ADP  2 consequences to impaired functioning of TCA cycle – most common situation leading to impaired function of

TCA cycle is relative lack of O2 to accept electrons in electron-transport chain o Inability to generate ATP from fuel oxidation o Accumulation of TCA cycle precursors . For example, inhibition of pyruvate oxidation in TCA cycle results in its reduction to lactate, which can cause lactic acidosis  Confirmation of suspected thiamine deficiency requires measuring thiamine levels; standard assay for determining if thiamine levels sufficient for metabolic processes is to use enzyme transketolase, which requires thiamine pyrophosphate (TPP) for activity o Transketolase can be obtained from RBCs, and measurement of transketolase activity done in absence and presence of exogenous TPP; if difference >25%, then thiamine deficiency confirmed o High-performance liquid chromatography (HPLC) analysis to quantify levels of both free thiamine and active form (TPP) in biologic fluids o Microbiologic assays measure bacterial cell growth in presence of sample that contains thiamine . Because bacteria require thiamine for growth, samples with more thiamine will stimulate more cell growth than samples that contain lesser levels of the vitamin Overview of TCA Cycle  Frequently called Krebs cycle or citric acid cycle (because citrate was one of first compounds known to participate); most common name is tricarboxylic acid cycle and denotes involvement of tricarboxylates citrate and isocitrate  In order for body to generate large amounts of ATP, major pathways of fuel oxidation generate acetyl-CoA, which is substrate of TCA cycle  First step of TCA cycle – acetyl portion of acetyl-CoA combines with 4-carbon intermediate oxaloacetate to form citrate (6 carbons), which is rearranged to form isocitrate  In next 2 oxidative decarboxylation reactions, electrons transferred to NAD+ to form NADH, and 2 molecules of

electron-depleted CO2 released; subsequently high-energy bond in GTP generated from substrate-level phosphorylation  Succinate oxidized to oxaloacetate with generation of one FAD(2H) and one NADH

 Net reaction of TCA cycle shows 2 carbons of acetyl group oxidized to 2 CO2, with conservation of energy as 3 NADH, 1 FAD(2H), and 1 GTP  TCA cycle requires large number of vitamins and minerals to function, including niacin (in NAD+), riboflavin (in FAD and FMN), pantothenic acid (in CoA), thiamine, Mg2+, Ca2+, Fe2+, and phosphate Reactions of TCA Cycle  Function of cycle is to conserve energy from oxidation, which it accomplishes principally by transferring electrons from intermediates of cycle to NAD+ or FAD o 8 electrons donated by acetyl group eventually end up in 3 NADH and 1 FAD(2H) o As consequence, ATP can be generated from oxidative phosphorylation when NADH and FAD(2H)

donate their electrons to O2 via electron-transport chain  Initially, acetyl group incorporated into citrate, an intermediate of TCA cycle; as citrate progresses through cycle to oxaloacetate, it is oxidized by 4 dehydrogenases (isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase), which remove electron-containing hydrogen or hydride atoms from substrate and transfer them to electron-accepting coenzymes (NAD+ or FAD)  Isomerase aconitase rearranges electrons in citrate, thereby forming isocitrate to facilitate electron transfer to NAD+ o Iron cofactor of aconitase facilitates isomerization

 Although no O2 introduced into TCA cycle, 2 molecules of CO2 produced have more oxygen than acetyl group;

oxygen atoms ultimately derived from carbonyl group of acetyl-CoA, 2 H2O added by fumarase and citrate 2- synthase, and PO4 added to GDP  Overall yield of energy-containing compounds from TCA cycle is 3 NADH, 1 FAD(2H), and 1 GTP o High-energy phosphate bond of GTP generated from substrate-level phosphorylation catalyzed by succinate thiokinase (succinyl-CoA synthetase) o As NADH and FAD(2H) reoxidized in electron-transport chain, about 2.5 ATP generated for each NADH and 1.5 ATP for FAD(2H)  Net energy yield from TCA cycle and oxidative phosphorylation is 10 high-energy bonds for each acetyl group oxidized

 Acetyl group of acetyl-CoA donates 8 electrons and 2 carbons – ultimate source of the carbons in the CO 2 produced and source of electrons in the FAD(2H) and NADH; same carbon atoms and electrons that enter from

one molecule of acetyl-CoA do not leave as CO2, NADH, FAD(2H) within same turn of cycle  Increasing rate of ATP utilization and fuel oxidation of TCA cycle (through exercise) causes rate of electron- transport chain to increase, and TCA cycle and other fuel oxidative pathways respond by increasing rates of NADH and FAD(2H) production Formation and Oxidation of Isocitrate  TCA cycle begins with condensation of activated acetyl group and oxaloacetate to form 6-carbon intermediate citrate (reaction catalyzed by citrate synthase) – (synthases in general catalyze condensation of 2 organic molecules to form carbon-carbon bond in absence of high-energy phosphate bond energy; catalyzes same type of reaction, but requires high-energy phosphate bonds to complete reaction) – because oxaloacetate regenerated with each turn of cycle, it is not considered substrate of cycle or source of electrons or carbon  Next step – hydroxyl group of citrate moved to adjacent carbon so it can be oxidized to form keto group; isomerization of citrate to isocitrate catalyzed by aconitase (named for intermediate of reaction) o Isocitrate dehydrogenase catalyzes oxidation of alcohol group and subsequent cleavage of carboxyl

group to release CO2 (oxidation followed by decarboxylation) to form α-ketoglutarate α-Ketoglutarate to Succinyl Coenzyme A  Next step of TCA cycle is oxidative decarboxylation of α-ketoglutarate to succinyl-CoA, catalyzed by α- ketoglutarate dehydrogenase complex o Dehydrogenase complex contains coenzymes TPP, lipoic acid, and FAD

 One of carboxyl groups of α-ketoglutarate released as CO2, and adjacent keto group oxidized to level of acid, which then combines with CoASH to form succinyl-CoA o Energy from reaction conserved principally in reduction state of NADH, with smaller amount present in high-energy thioester bond of succinyl-CoA Generation of Guanosine Triphosphate

 Energy from succinyl-CoA thioester bond used to generate GTP from GDP and inorganic phosphate (P i) in reaction catalyzed by succinate thiokinase (succinyl-CoA synthetase in reverse reaction)  Reaction is example of substrate-level phosphorylation (formation of high-energy phosphate bond where none

previously existed without use of molecular O2 [not oxidative phosphorylation])  High-energy phosphate bond of GTP energetically equivalent to that of ATP and can be used directly for energy- requiring reactions such as protein synthesis Oxidation of Succinate to Oxaloacetate +  Transfer 2 pairs of electrons to FAD and NAD and add H2O, regenerating oxaloacetate  Sequence of reactions that converts succinate to oxaloacetate begins with oxidation of succinate to fumarate o Single electrons transferred from 2 adjacent –CH2– methylene groups of succinate to FAD bound to succinate dehydrogenase, thereby forming double bond of fumarate o From reduced enzyme-bound FAD, electrons passed into electron-transport chain  OH- group and proton from water add to double bond of fumarate, converting it to malate  In last reaction of TCA cycle, alcohol group of malate oxidized to keto group through donation of electrons to NAD+  TCA cycle complete – chemical bond energy, carbon, and electrons donated by acetyl group have been

converted to CO2, NADH, FAD(2H), GTP, and heat  Succinate-to-oxaloacetate sequence of reactions (oxidation through formation of double bond, addition of water to double bond, and oxidation of resultant alcohol to ketone) found in many oxidative pathways in cell, such as pathways for oxidation of fatty acids and oxidation of BCAAs  Coenzymes FAD and FMN synthesized from vitamin riboflavin, which is actively transported into cells where enzyme flavokinase adds phosphate to form FMN; FAD synthetase adds AMP to form FAD, which is major coenzyme in tissues and generally found tightly bound to proteins (10% covalently bound) o FAD’s turnover in body is very slow, and people can live for long periods on low intakes without displaying any signs of riboflavin deficiency

 Isocitrate dehydrogenase releases first CO2, and α-ketoglutarate dehydrogenase releases second CO2; no net consumption of oxaloacetate in TCA cycle (first step uses oxaloacetate and last step produces one) Coenzymes of Tricarboxylic Acid Cycle  Enzymes of TCA cycle rely heavily on coenzymes for catalytic function o Isocitrate dehydrogenase and malate dehydrogenase use NAD+ as coenzyme o Succinate dehydrogenase uses FAD o Citrate synthase catalyzes reaction that uses CoA derivative (acetyl-CoA) o α-ketoglutarate dehydrogenase complex uses TPP, lipoate, and FAD as bound coenzymes, and NAD+ and CoASH as substrates  FAD able to accept single electrons (H·) and forms half-reduced single-electron intermediate, so participates in reactions in which single electrons transferred independently from 2 different atoms, which occurs in double- bond formation (succinate to fumarate) and disulfide bond formation (lipoate to lipoate disulfide in α- ketoglutarate dehydrogenase reaction)  NAD+ accepts pair of electrons as H-, which is attracted to carbon opposite positively charged pyridine ring; occurs in oxidation of alcohols to ketones by malate dehydrogenase and isocitrate dehydrogenase o Nicotinamide ring accepts H- from C-H bond, and alcoholic H released into medium as H+  Free radical, single-electron forms of FAD very reactive, and FADH can lose its electron through exposure to water or initiation of chain reactions, so FAD must remain very tightly, sometimes covalently, attached to its enzyme while it accepts and transfers electrons to another group bound on enzyme o Because FAD interacts with many functional groups on AA side chains in active site, E0’ for enzyme- bound FAD varies greatly and can be greater or much less than that of NAD+  NAD+ and NADH more like substrate and product than coenzymes  Succinate oxidation cannot produce energy without oxygen; succinate oxidized to fumarate by donation of

electrons to FAD; however, ATP can be generated from this process only when electrons donated to O2 in electron-transport chain o Energy generated by electron-transport chain used for ATP synthesis in process of oxidative phosphorylation; after covalently bound FAD(2H) oxidized back to FAD by electron-transport chain, succinate dehydrogenase can oxidize another succinate molecule  NADH plays regulatory role in balancing energy metabolism that FAD(2H) cannot because FAD(2H) remains attached to enzyme o Free NAD+ binds to a dehydrogenase and is reduced to NADH that is then released into medium where it can bind and inhibit a different dehydrogenase o Oxidative enzymes controlled by NADH/NAD+ ratio and do not generate NADH faster than it can be reoxidized in electron-transport chain o Regulation of TCA cycle and other pathways of fuel oxidation by NADH/NAD+ ratio part of mechanism for coordinating rate of fuel oxidation to rate of ATP use  CoASH – acylation coenzyme; participates in reactions through formation of thioester bond between S of CoASH and an acyl group (acetyl-CoA, succinyl-CoA, etc.) o Thioester bond differs from typical oxygen ester bond because S, unlike O, does not share electrons and participate in resonance formations . One of consequences of this is that carbonyl carbon, α-carbon, and β-carbon of acyl group in CoA thioester can be activated for participation in different types of reactions (in citrate synthase reaction, α-carbon methyl group activated for condensation with oxaloacetate) . Another consequence is thioester bond is high-energy bond that has large negative ΔG0’ of hydrolysis o Energy from cleavage of high-energy thioester bonds of succinyl-CoA and acetyl-CoA used in 2 different ways in TCA cycle . When succinyl-CoA thioester bond cleaved by succinate thiokinase, energy used directly for activating enzyme-bound phosphate that is transferred to GDP . When thioester bond of acetyl-CoA cleaved in citrate synthase reaction, energy released, giving reaction a large negative ΔG0’, which helps keep TCA cycle going in forward direction o CoASH synthesized from vitamin pantothenate in sequence of reactions that phosphorylate pantothenate, add sulfhydryl portion of CoA from cysteine, and then add AMP and an additional phosphate group from ATP . Pantothenate widely distributed in foods, so very unlikely to develop a deficiency . Although CoA required in approximately 100 different reactions, no recommended dialy allowance has been established for pantothenate because indicators have not yet been found that specifically and sensitivetly reflect deficiency of this vitamin in a human . Reported symptoms of pantothenate deficiency (fatigue, nausea, and loss of appetite) characteristic of vitamin deficiencies in general  α-ketoglutarate dehydrogenase complex is one of 3-member family of similar α-keto acid dehydrogenase complexes (other members are pyruvate dehydrogenase complex [PDC] and BCAA α-keto acid dehydrogenase complex) – each complex specific for different α-keto acid structure o In sequence of events catalyzed by complexes, α-keto acid decarboxylated (releases carboxyl group as

CO2), and keto group oxidized to level of carboxylic acid and then combined with CoASH to form acyl- CoA thioester (for example, succinyl-CoA) o All α-keto acid dehydrogenase complexes are huge enzyme complexes composed of multiple subunits of

3 different enzymes, designated E1, E2, and E3

. E1 is α-keto acid decarboxylase that contains TPP; cleaves off carboxyl group of α-keto acid

. E2 is transacylase-containing lipoate; transfers acyl portion of α-keto acid from thiamine to CoASH

. E3 is dihydrolipoyl dehydrogenase, which contains FAD; transfers electrons from reduced lipoate to NAD+ o Collection of 3 enzyme activities into one huge complex enables product of one enzyme to be transferred to next enzyme without loss of energy; complex formation also increases rate of catalysis

because substrates for E2 and E3 remain bound to enzyme complex o Heart failure can be cause by dietary deficiency of thiamine; pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and branched-chain α-keto acid dehydrogenase complexes less functional than normal . Because heart muscle, skeletal muscle, and nervous tissue have high rates of ATP production

from NADH produced by oxidation of pyruvate to acetyl-CoA and of acetyl-CoA to CO2 in TCA cycle, these tissues present with most obvious signs of thiamine deficiency . In Western societies, gross thiamine deficiency most often associated with alcoholism; mechanism for active absorption of thiamine strongly and directly inhibited by alcohol . Subclinical deficiency of thiamine from malnutrition or anorexia may be common in general population and usually associated with multiple vitamin deficiencies o TPP synthesized from thiamine by addition of pyrophosphate, which binds Mg, which binds to AA side chains on enzyme; binding is relatively weak for a coenzyme, so thiamine turns over rapidly in body, and deficiency can develop rapidly in individuals on low-thiamine diets . General function of TPP is cleavage of C-C bond next to keto group; in α-ketoglutarate, pyruvate, and branched-chain α-keto acid dehydrogenase complexes, functional carbon on thiazole ring forms covalent bond with α-keto carbon, thereby cleaving bond between α-keto carbon and adjacent carboxylic acid group . TPP also coenzyme for transketolase in pentose phosphate pathway, where it cleaves C-C bond next to keto group . In thiamine deficiency, α-ketoglutarate, pyruvate, and other α-keto acids accumulate in blood o Lipoate – coenzyme found only in α-keto acid dehydrogenase complexes; synthesized rom carbohydrate and AAs and does not require a vitamin precursor . Attached to transacylase enzyme through its carboxyl group, which is covalently bound to

terminal -NH2 of lysine in the protein . At functional end, lipoate contains disulfide group that accepts electrons when it binds to acyl fragment from thiamine and transfer it to active site containing bound CoASH . Lipoate then swings over to dihydrolipoyl dehydrogenase to transfer electrons from lipoyl sulfhydryl groups to FAD o FAD on dihydrolipoyl dehydrogenase accepts electrons from lipoyl sulfhydryl groups and transfers them to bound NAD+; FAD accepts and transfers electrons without leaving binding site on enzyme . Direction of reaction favored by interactions of FAD with groups on enzyme, which change reduction potential, and by overall release of energy from cleavage and oxidation of α- ketoglutarate  Arsenic poisoning caused by presence of large number of different arsenious compounds that are effective metabolic inhibitors 2- 2- o Acute arsenic poisoning requires high doses and involves arsenate (AsO4 ) and arsenite (AsO3 ) o Arsenite 10x more toxic than arsenate; binds to neighboring sulfhydryl groups, such as those in dihydrolipoate and in nearby cysteine pairs (vicinal) found in α-keto acid dehydrogenase complexes and in succinic dehydrogenase o Arsenate weakly inhibits enzymatic reactions involving phosphate, including enzyme glyceraldehyde 3-P dehydrogenase in glycolysis . Both aerobic and anaerobic ATP production can be inhibited o Low doses of arsenic compounds in water more associated with increased risk of cancer than direct toxicity Energetics of TCA Cycle  TCA cycle operates with overall net negative ΔG0’; conversion of substrates to products energetically favorable o Some reactions, such as malate dehydrogenase reaction, have positive value 0 o Net ΔG ’ of reaction lost as heat (amount of energy spent to ensure oxidation of acetyl group to CO2 goes to completion) o Oxidation of NADH and FAD(2H) in electron-transport chain help make acetyl oxidation more energetically favorable and pulls TCA cycle forward  Reactions of TCA cycle extremely efficient in converting energy in chemical bonds of acetyl group to other forms o TCA cycle reactions able to conserve about 90% of energy available from oxidation of acetyl-CoA  Three reactions of TCA cycle have large negative ΔG0’s that strongly favor forward direction: reactions catalyzed by citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase o Within TCA cycle, these reactions physiologically irreversible because products do not rise to high enough concentrations under physiologic conditions to overcome large negative ΔG0’ values and enzymes involved catalyze reverse reaction very slowly o These reactions make major contribution to overall negative ΔG0’ for TCA cycle and keep it going in forward direction  Reactions catalyzed by aconitase and malate dehydrogenase have positive ΔG0’ for forward direction and are thermodynamically and kinetically reversible o Because aconitase is rapid in both directions, equilibrium values for concentration ratio of products to substrates is maintained, and concentration of citrate is 20x that of isocitrate . Accumulation of citrate instead of isocitrate facilitates transport of excess citrate to cytosol, where it can provide source of acetyl-CoA for pathways such as fatty acid and cholesterol synthesis . Allows citrate to serve as inhibitor of citrate synthase when flux through isocitrate dehydrogenase decreased o Equilibrium constant of malate dehydrogenase reaction favors accumulation of malate over oxaloacetate, resulting in low oxaloacetate concentration influenced by NADH/NAD+ ratio . Net flux of oxaloacetate toward malate in liver during fasting (as result of fatty acid oxidation, which raises NADH/NAD+ ratio), and malate can then be transported out of mitochondria to provide substrate for gluconeogenesis  Those out of shape can have difficulty losing weight because human fuel utilization too efficient o Adipose tissue fatty acids being converted to acetyl-CoA, which is being oxidized in TCA cycle, thereby generating NADH and FAD(2H), and energy from them used for ATP synthesis from oxidative phosphorylation o If fuel utilization were less efficient and ATP yield was lower, they could oxidize much greater amounts of fat to get ATP they need for exercise Regulation of TCA Cycle  Oxidation of acetyl-CoA in TCA cycle and conservation of energy as NADH and FAD(2H) essential for generation of ATP in almost all tissues in body o In spite of changes in supply of fuels, type of fuels in blood, or rate of ATP utilization, cells maintain ATP homeostasis o Rate of TCA cycle principally regulated to correspond to rate of electron-transport chain, which is regulated by ATP/ADP ratio and rate of ATP utilization  2 major messengers feed info on rate of ATP utilization back to TCA cycle o Phosphorylation state of ATP, as reflected in ATP and ADP levels o Reduction state of NAD+, as reflected in ratio of NADH/NAD+  In cell or mitochondrion, total adenine nucleotide pool (AMP, ADP, and ATP) and total NAD pool (NAD+ and NADH) relatively constant  As someone exercises, myosin ATPase hydrolyzes ATP to provide energy for movement of myofibrils o Decrease of ATP and increase of ADP stimulates electron-transport chain to oxidize more NADH and FAD(2H); TCA cycle stimulated to provide more NADH and FAD(2H) to electron-transport chain o Activation of TCA cycle occurs through decrease of NADH/NAD+ ratio, increase of ADP concentration, and increase of Ca2+ o Regulation of transcription of genes for TCA cycle enzymes too slow to respond to changes of ATP demands during exercise, but number and size of mitochondria increase during training, so one can increase capacity for fuel oxidation as they train  In pathways subject to feedback regulation, first step of pathway must be regulated so precursors flow into alternative pathways if product not needed o Citrate synthase (first enzyme of TCA cycle) is simple enzyme that has no allosteric regulators; its rate controlled principally by concentration of oxaloacetate (its substrate) and concentration of citrate (product inhibitor that is competitive with oxaloacetate) o Malate-oxaloacetate equilibrium favors malate, so oxaloacetate concentration very low inside

mitochondrion and is below apparent Km of citrate synthase o When NADH/NAD+ ratio decreases, ratio of oxaloacetate to malate increases o When isocitrate dehydrogenase activated, concentration of citrate decreases, thus relieving product inhibition of citrate synthase o Both increased oxaloacetate and decreased citrate levels regulate response of citrate synthase to conditions established by electron-transport chain and oxidative phosphorylation o In liver, NADH/NAD+ ratio helps determine whether acetyl-CoA enters TCA cycle or goes into alternative pathway for ketone body synthesis  Regulation of pathway can occur by regulating enzyme that catalyzes rate-limiting step o Isocitrate dehydrogenase (one of rate-limiting steps of TCA cycle) allosterically activated by ADP and inhibited by NADH; in absence of ADP, enzyme exhibits positive cooperativity (as isocitrate binds to one subunit, other subunits converted to active conformation) . In presence of ADP, all subunits in active conformation, and isocitrate binds more readily, so

apparent Km (S0.5) shifts to much lower value . At concentration of isocitrate found in mitochondrial matrix, small change in concentration of ADP can produce large change in rate of isocitrate dehydrogenase reaction . Small changes in concentration of NADH and NAD+ also affect rate of enzyme more than they would a nonallosteric enzyme  α-ketoglutarate dehydrogenase complex (not an allosteric enzyme) is product inhibited by NADH and succinyl- CoA and may be inhibited by GTP o Both α-ketoglutarate dehydrogenase and isocitrate dehydrogenase respond directly to changes in relative levels of ADP and rate at which NADH oxidized by electron transport o Both enzymes also activated by Ca2+; in contracting heart muscle, release of Ca2+ from sarcoplasmic reticulum provides additional activation of enzymes when ATP being rapidly hydrolyzed  Regulation of TCA cycle ensures NADH generated fast enough to maintain ATP homeostasis and regulates concentration of TCA cycle intermediates o In liver, decreased rate of isocitrate dehydrogenase increases citrate concentration, which stimulates citrate efflux to cytosol o Several regulatory interactions control levels of TCA intermediates and their flux into pathways that adjoin TCA cycle Precursors of Acetyl-CoA  Compounds enter TCA cycle as acetyl-CoA or as intermediate that can be converted to malate or oxaloacetate;

compounds that enter as acetyl-CoA oxidized to CO2, and those that enter as intermediates replenish intermediates that have been used in biosynthetic pathways, such as gluconeogenesis or heme synthesis, but

cannot be fully oxidized to CO2  Acetyl-CoA serves as common point of convergence for major pathways of fuel oxidation; generated directly from β-oxidation of fatty acids and degradation of ketone bodies β-hydroxybutyrate and acetoacetate o Formed form acetate, which can arise from diet or from ethanol oxidation o Glucose and other carbohydrates enter glycolysis and are oxidized to pyruvate o Alanine and serine converted to pyruvate o Pyruvate oxidized to acetyl0CoA by PDC o Several AAs such as leucine and isoleucine oxidized to acetyl-CoA

o Final oxidation of acetyl-CoA to CO2 in TCA cycle is last step in all major pathways of fuel oxidation  PDC oxidizes pyruvate to acetyl-CoA, linking glycolysis and TCA cycle; in brain, which is dependent on oxidation

of glucose to CO2 to fulfill ATP needs, regulation of PDC is life-and-death matter o PDC belongs to α-keto acid dehydrogenase complex family and shares structural and catalytic features with α-ketoglutarate dehydrogenase complex and branched-chain α-keto acid dehydrogenase complex . Contains pyruvate decarboxylase subunits that bind TPP, transacetylase subunits that bind lipoate, and dihydrolipoyl dehydrogenase subunits that bind FAD

. Although E1 and E2 enzymes in PDC relatively specific for pyruvate, same dihydrolipoyl dehydrogenase participates in all α-keto acid dehydrogenase complexes

. PDC complex contains E3 binding protein (E3-BP) . Each functional component of PDC complex present in multiple copies

. E1 enzyme is a tetramer of α and β subunits o Deficiencies of PDC among most common inherited diseases leading to lactic acidemia and are grouped into category of Leigh disease (subacute necrotizing encephalopathy) . In severe form, PDC deficiency presents with overwhelming lactic acidosis at birth, with death in neonatal period . In moderate form, lactic acidemia moderate, but there is profound psychomotor retardation with increasing age . In many cases, concomitant damage to brainstem and basal ganglia lead to death in infancy . Neurologic symptoms arise because brain has very limited ability to use fatty acids as fuel and is therefore dependent on glucose metabolism for energy

. Most common PDC genetic defects are in gene for α-subunit of E1 (X-linked dominant) . Because of its importance in CNS metabolism, pyruvate dehydrogenase deficiency is problem in both males and females, even if female is only carrier o PDC activity controlled principally through phosphorylation by pyruvate dehydrogenase kinase, which inhibits enzyme and dephosphorylation by pyruvate dehydrogenase phosphatase, which activates it . Pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase are regulatory subunits in PDC complex and act only on complex  PDC kinase transfers phosphate from ATP to specific serine hydroxyl (ser-OH) gropus on

pyruvate decarboxylase (E1)  PDC phosphatase removes phosphate groups by hydrolysis

. Phosphorylation of just one serine in PDC E1 α-subunit can decrease activity by >99% . PDC kinase present in complexes as tissue-specific isozymes that vary in regulatory properties . PDC kinase inhibited by ADP and pyruvate, so when rapid ATP utilization results in increase of ADP or when activation of glycolysis increases pyruvate levels, PDC kinase inhibited and PDC remainse active, nonphosphorylated form . PDC phosphatase requires Ca2+ for full activity; in heart, increased intramitochondrial Ca2+ during rapid contraction activates phosphatase, thereby increasing amount of active, nonphosphorylated PDC . PDC regulated through inhibition by products (acetyl-CoA and NADH); this inhibition stronger than regular product inhibition because binding to PDC stimulates its phosphorylation to inactive form  CoASH and NAD+ antagonize product inhibition  When ample supply of acetyl-CoA for TCA cycle already available from fatty acid oxidation, acetyl-CoA and NADH build up and dramatically decrease further synthesis by PDC . PDC activated rapidly through mechanism involving insulin, which plays prominent role in adipocytes  In many tissues, insulin may over time slowly increase amount of PDC present  Rate of other fuel oxidation pathways that feed into TCA cycle increased when ATP utilization increases  Insulin, other hormones, and diet – control availability of fuels for oxidative pathways TCA Cycle Intermediates and Anaplerotic Reactions  Intermediates of TCA cycle serve as precursors for variety of different pathways present in different cell types; particularly important in central metabolic role of liver o TCA cycle in liver often called open cycle because there is such high efflux of intermediates o After high-carb meal, citrate efflux and cleavage to acetyl-CoA provides acetyl units for cytosolic fatty acid synthesis o During fasting gluconeogenic precursors converted to malate, which leaves mitochondria for cytosolic gluconeogenesis o Liver uses TCA cycle intermediates to synthesize carbon skeletons of AAs  Succinyl-CoA may be removed from TCA cycle to form heme in cells of liver and bone marrow  In brain, α-ketoglutarate converted to glutamate and then to GABA (neurotransmitter)  In skeletal muscle, α-ketoglutarate converted to glutamine, which is transported through blood to other tissues  Pyruvate, citrate, α-ketoglutarate, malate, ADP, ATP, and phosphate (as well as many other compounds) have specific transporters in inner mitochondrial membrane that transport compounds between mitochondrial matrix and cytosol in exchange for compound of similar charge  CoASH, acetyl-CoA, other CoA derivatives, NAD+, NADH, and oxaloacetate are not transported at metabolically significant rate o To obtain acetyl-CoA, many cells transport citrate to cytosol, where it is cleaved to acetyl-CoA and oxaloacetate by citrate lyase  Removal of any intermediates from TCA cycle removes 4 carbons used to regenerate oxaloacetate during each turn of cycle; with depletion of oxaloacetate, it is impossible to continue oxidizing acetyl-CoA; to enable TCA cycle to keep running, cells have to supply enough 4-carbon intermediates from degradation of carbs or certain AAs to compensate for rate of removal – pathways or reactions that replenish intermediates of TCA cycle are anaplerotic (filling up)

o Pyruvate carboxylase – one of major anaplerotic enzymes; catalyzes addition of CO2 to pyruvate to form oxaloacetate

. Pyruvate carboxylase contains biotin, which forms covalent intermediate with CO2 in reaction 2+ that requires ATP and Mg ; activated CO2 transferred to pyruvate to form carboxyl group of oxaloacetate . Found in liver, brain, adipocytes, and fibroblasts, where its function is anaplerotic; concentration high in liver and kidney cortex, where there is continuous removal of oxaloacetate and malate from TCA cycle to enter gluconeogeneic pathway . Activated by acetyl-CoA and inhibited by high concentrations of many acyl-CoA derivatives . As concentration of oxaloacetate depleted through efflux of TCA cycle intermediates, rate of citrate synthase reaction decreases and acetyl-CoA concentration rises; acetyl-CoA then activates pyruvate carboxylase to synthesize more oxaloacetate . Pyruvate carboxylase deficiency – one of genetic diseases grouped under clinical manifestations of Leigh disease; in mild form, patient presents early in life with delayed development and mild- to-moderate lactic acidemia; patients who survive severely mentally retarded and there is loss of cerebral neurons  In brain, pyruvate carboxylase present in astrocytes, which use TCA cycle intermediates to synthesize glutamine – essential for neuronal survival  Major cause of lactic acidemia is cells dependent on pyruvate carboxylase for anaplerotic supply of oxaloacetate cannot oxidize pyruvate in TCA cycle because of low oxaloacetate levels, and liver cannot convert pyruvate to glucose because pyruvate carboxylase reaction required for pathway to continue, so excess pyruvate converted to lactate o Amino acid degradation – pathways for oxidation of many AAs convert carbon skeletons into 5- and 4- carbon intermediates of TCA cycle that can regenerate oxaloacetate . Alanine and serine carbons enter through pyruvate carboxylase . In all tissues with mitochondria (except for liver), oxidation of 2 BCAAs isoleucine and valine to succinyl-CoA forms major anaplerotic route . In liver, other compounds that form propionyl-CoA (methionine, threonine, and odd-chain- length or branched fatty acids) also enter TCA cycle as succinyl-CoA . In most tissues, glutamine taken up from blood, converted to glutamate, and then oxidized to α- ketoglutarate, forming another major anaplerotic route . TCA cycle can’t be resupplied with intermediates by even-chain-length fatty acid oxidation or by ketone body oxidation, which forms only acetyl-CoA . In TCA cycle, 2 carbons lost from citrate before succinyl-CoA formed, so no net conversion of acetyl carbon to oxaloacetate Compartmentation of Mitochondrial Enzymes  Mitochondrion forms structural, functional, and regulatory compartment within cell o Inner mitochondrial membrane impermeable to ions, and compounds can cross membrane only on specific transport proteins o Enzymes of TCA cycle have more direct access to products of previous reaction in pathway than they would if products were able to diffuse throughout cell o Complex formation between enzymes restricts access to pathway intermediates o Malate dehydrogenase and citrate synthase may form loosely associated complex o Multienzyme pyruvate dehydrogenase and α-ketoglutarate dehydrogenase complexes examples of substrate channeling by tightly bound enzymes . Only transacylase enzyme has access to thiamine-bound intermediate of reaction, and only lipoamide dehydrogenase has access to reduced lipoic acid  Compartmentation plays important role in regulation – close association between rate of electron-transport chain and rate of TCA cycle maintained by mutual access to same pool of NADH and NAD+ in mitochondrial matrix o NAD+, NADH, CoASH, and acyl-CoA derivatives have no transport proteins and can’t cross mitochondrial membrane, so all dehydrogenases compete for same NAD+ molecules and inhibited when NADH rises o Accumulation of acyl-CoA derivatives in mitochondrial matrix affects other CoA-using reactions, either by competing at active site or limiting CoASH availability Import of Nuclear-Encoded Proteins  All mitochondrial matrix proteins, such as TCA cycle enzymes, encoded by nuclear genome and are imported into mitochondrial matrix as unfolded proteins that are pushed and pulled through channels in outer and inner mitochondrial membranes  Proteins destined for mitochondrial matrix have either targeting N-terminal presequence that includes several positively charged AA residues or internal mitochondrial localizing signal  Mitochondrial matrix proteins synthesized on free ribosomes in cytosol and maintain unfolded conformation by binding hsp70 chaperonins  Basic presequence binds to receptor in translocases of outer membrane (TOM) complex, which consists of channel proteins, assembly proteins, and receptor proteins with different specificities (e.g., TOM20 binds matrix protein presequence) o Negatively charged acidic residues on receptors and in channel pore assist in translocation of matrix protein through channel, presequence first  Matrix preprotein translocated across inner membrane through translocases of inner membrane (TIM) complex; insertion of preprotein into TIM channel driven by potential difference across membrane (Δψ) o Mitochondrial hsp70 (mthsp70 – bound to matrix side of TIM complex) binds incoming preprotein and may ratchet it through membrane o ATP required for binding of mthsp70 to TIM complex and for subsequent dissociation of mthsp70 and matrix preprotein  In matrix, preprotein may require another heat-shock protein (hsp60) for proper folding  Final step in import process is cleavage of signal sequence by matrix-processing protease  Proteins of inner mitochondrial membrane imported through similar process, using TOM and Tim complexes while containing different protein components