Lecture 3- TCA Cycle PDF

Tricarboxylic acid (TCA) cycle Prof. Dr. Gerhard Grüber Nanyang Technological University School of Biological Sciences [email protected]  Energy transformations in fuel metabolism When ATP energy is transformed into cellular responses, such as muscle contraction, ATP is...

Tricarboxylic acid (TCA) cycle Prof. Dr. Gerhard Grüber Nanyang Technological University School of Biological Sciences [email protected]  Energy transformations in fuel metabolism When ATP energy is transformed into cellular responses, such as muscle contraction, ATP is cleaved to ADP and Pi. In cellular respiration, O2 is In phase 1 of respiration, energy is conserved from fuel oxidation used for regenerating ATP from by enzymes that transfer electrons from the fuels to the electron- oxidation of fuels to CO2. accepting coenzymes nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD), which are reduced to NADH and FAD(2H), respectively. The pathways for the oxidation of most fuels (glucose, fatty acids, ketone bodies, and many amino acids) converge in the generation of the activated 2-carbon acetyl group in acetyl coenzyme A (acetyl- CoA). The complete oxidation of the acetyl group to CO2 occurs in the tricarboxylic acid (TCA) cycle, which collects the energy mostly as NADH and FAD(2H). C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Overview of glycolysis and important reactions We call ATP a high energy compound and mean that it contains at least one bond with a sufficiently favorable ∆G° of hydrolysis. Calling a substance a high-energy compound does not mean that it is chemically unstable or unusually reactive. ATP is kinetically stable - its spontaneous hydrolysis is slow at physiological pH and temperature. ATP breakdown is usually coupled with a thermodynamically unfavorable reaction, such as the synthesis of glucose- 6-phosphate from glucose. Thus, it is more accurate to say that ATP has a high phosphoryl group transfer potential. The hexokinase reaction involves a nucleophilic attack of the C6-OH of glucose on the electrophilic terminal γ-phosphate of ATP. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition D.R. Appling, S.J. Anthony-Cahill & C.K. Mathews, Biochemistry Concepts & Connections, 2nd edition  Oxidative Fates of Pyruvate and Nicotinamide Adenine Dinucleotide Remember: Carbon atoms become oxidized either through loss of a hydride ion (H - ) or through combination with oxygen as we will see in the respiratory chain. When an organic compound loses a hydride ion, it loses both shared electrons associated with the C-H bond. Because most metabolic oxidations involve loss of hydrogen (typically a hydride ion H - , plus a proton, H + ) from the electron donor, we call enzymes that catalyze those reactions dehydrogenases, C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition D. R. Appling, S. Anthony-Cahill, C. K. Mathews, Biochemistry: Concepts and Connections, 2nd edition (2019)  Anaerobic conversion of Pyruvate to Ethanol Oxidative decarboxylate The synthesis of ethanol by highly selected strains of yeast is important in the production of beer and wine. Yeast cells convert pyruvate to ethanol and CO2 and oxidize NADH to NAD+. Two reactions are required: Pyruvate is decarboxylated in an irreversible reaction catalyzed by pyruvate decarboxylase. Alcoholdehydrogenase catalyzes the reduction of acetaldehyde to ethanol. This oxidation-reduction reaction is coupled to oxidation of NADH. The sum of the glycolytic reaction and the conversion of pyruvate to ethanol is L. A. Moran, H. R. Horton, K. G. Scrimgeour & M. D. Perry, Principles in Biochemistry, 5th edition D. L. Nelson, M. & M. Cox, Lehninger Principles of Biochemistry, 4th edition  The citric acid cycle and what we will learn today Citric acid cycle also called Krebs cycle or Tricarboxyl Acid cycle (TCA) Connection between glycolysis and the TCA cycle: Pyruvate dehydrogenase complex TCA cycle reactions Regulation of the TCA cycle provide precusers ~ TCA cycle is an amphibolic pathway for other pathway Anaplerotic (Filling-Up) reactions & receive intermediates from other pathways  The loci of Substrate-level phosphorylation Electrons Electrons via NADH via NADH and FADH2 GLYCOLYSIS PYRUVATE OXIDATION CITRIC ACID Glucose Pyruvate Acetyl CoA CYCLE CYTOSOL MITOCHONDRION ATP ATP Substrate-level Substrate-level © 2017 Pearson Education, Ltd  Convertion of pyruvate to acetyl-CoA by oxidative decarboxylation DGo’ = -31.5 KJ/mol Lexergovic extE) The Pyruvate Dehydrogenase Multienzyme Complex (PDC) is a cluster of three enzymes: E1, pyruvate dehydrogenase; E2, dihydrolipoyl transacetylase; E3, dihydrolipoyl dehydrogenase They require 5 cofactors: thiamine pyrophosphate (TPP), Lipoic acid, coenzyme A, flavin adenine dinucleotide (FAD), and NAD+. The reaction occurs in the mitochondrial matrix in eukaryotic cells.  Acetyl-coenzyme A also called Acetyl-CoA Functional role: acetyl group carrier (transfer). of ⚫ ~ trsf this gup ⚫ Reactive group: thiol (─SH). from pyruvate to ⚫ Acetyl group is covalently linked to the thiol Cot group forming a high-energy thioester. The & high-energy DGo’ for hydrolysis of its thioester bond is thioester -31.5 KJ/mol, which makes this reaction slightly (1 KJ/mol) more exergonic than that of ATP hydrolysis. The formation of this thioester bond in a metabolic intermediate conserves a portion of the free energy of oxidation of a metabolic fuel. ⚫ High free energy is released upon hydrolysis of the thioester linkage. That is why it has a high acyl group transfer potential. ⚫ CoA is the common product of carbohydrate-, ( fatty acid- and amino acid breakdown. phosphodiester bond Never forget me! Voet, Voet: BIOCHEMISTRY 3rd edition  Comparison of free energies of hydrolysis of thioesters and oxygen esters -good leaving grp I weaker bond Lack of resonance stabilization in thioesters is the basis for the more favorable ΔG of hydrolysis of thioesters, relative to that of ordinary oxygen esters. Most esters have two resonance forms. Stabilization involves π-electron orbital overlap, giving partial double-bond character to the C-OR link. This lack of double-bond character in the SR bond of acyl-CoAs makes this bond weaker than the corresponding C-OR bond in ordinary esters, and the thiolalkoxide ion (R-S - ) is a good leaving group in nucleophilic displacement reactions. Thus, the acyl group is readily transferred to other metabolites, which is what occurs in the first reaction of the citric acid cycle. D. R. Appling, S. Anthony-Cahill, C. K. Mathews, Biochemistry: Concepts and Connections, 2nd edition (2019)  Convertion of pyruvate to acetyl-CoA by oxidative decarboxylation DGo’ = -31.5 KJ/mol The Pyruvate Dehydrogenase Multienzyme Complex (PDC) is a cluster of three enzymes: E1, pyruvate dehydrogenase; E2, dihydrolipoyl transacetylase; E3, dihydrolipoyl dehydrogenase They require 5 cofactors: thiamine pyrophosphate (TPP), Lipoic acid, coenzyme A, flavin adenine dinucleotide (FAD), and NAD+. The reaction occurs in the mitochondrial matrix in eukaryotic cells.  Structural organization of the E. coli PDC all in closeanimity muisubunits helps for acctyl cort (a) The dihydrolipoyl transacetylase (E2) “core”. Its 24 subunits associate as trimers located at the corners of a cube to form a particle that has cubic symmetry. (b) The 24 pyruvate dehydrogenase (E1) subunits form dimers that associate with the E2 core (shaded cube) at the centre of each of its 12 edges, whereas the 12 dihydrolipoyl dehydrogenase (E3) ~ subunits form dimers that attach to the E2 cube at the centers of each of its 6 faces. a midenerated (b) Parts of a and b combined to form the entire 60-subunits complex. Voet, Voet: BIOCHEMISTRY 3rd edition  The coenzymes and prosthetic groups of PDC reducedpoic acid Voet, Voet: BIOCHEMISTRY 3rd edition  Interconversion of lipoamide and dihydrolipoamide um [H] acetyl grp bound to this Voet, Voet: BIOCHEMISTRY 3rd edition D. R. Appling, S. Anthony-Cahill, C. K. Mathews, Biochemistry: Concepts and Connections, 2nd edition (2019)  Thiamine pyrophosphate (TPP) ⚫ Pyrophosphate of thiamine (vitamin B1) ⚫ Function: transfer of an activated aldehyde unit ⚫ Functional group is the thiazolium ring, which has an acidic proton at C2. Loss of proton produces a carbanion, which is the active species. ⚫ Reaction is catalyzed by pyruvate dehydrogenase (E1) Voet, Voet: BIOCHEMISTRY 3rd edition  TPP in the pyruvate dehydrogenase reaction protonate (reduced ↑ 1 Nucleophilic attack by the ylid form of & undergo TPP on the carbonyl carbon of pyruvate 2 Departure of CO2 and formation of 1 Thiazolium ring : Catalytic rxt 4 hydroxyethyl TPP hydroxyethyl lipoamide on the arm swing from E -En 3 Transfer of the hydroxyethyl group to. us E2 the lipoamide moiety, occurring via an 2 attack of the hydroxyethyl carbanion on the lipoamide disulfide, followed by 3 & it swing to Es elimination of TPP to form an acetyl me thioester on dihydrolipoamide and regenerate E1. E, I 5 4 Transfer of the acetyl group to CoA. This nucleophilic acyl substitution reaction exchanges one thioester for another, giving acetyl-CoA and Es dihydrolipoamide. 5 The last two steps of the process are required to reoxidize the dihydrolipoamide of E2, and transfer the pair of electrons via FAD to NAD+. Karlson: Biochemistry 13th edition  Molecular formula and reactions of flavin adenine dinucleotide bound ~ covalently impt for e-trsf Important: The oxidized form, FAD is yellow with a λmax = 450 nm peak observed Never forget me! I 5 Important: The reduced form, FADH2 is colorless Voet, Voet: BIOCHEMISTRY 3rd edition  Structures and reaction of NAD+ and NADP+ loxidised) 200 nm UV-spectrum of NAD+ () and ~ NADH (○), respectively add =~34oum (reduced Voet, Voet: BIOCHEMISTRY 3rd edition Karlson: Biochemistry 13th edition  Summary of the Pyruvate dehydrogenase complex reaction 1. Pyruvate reacts with TPP, undergoes decarboxylation and forms hydroxyethyl-TPP of E1. 2. The aldehyde group is transferred to one lipoamide cofactor of E2 and simultaneously oxidized to acetyl. 3. The acetyl group is transferred to the next lipoamide cofactor. 4. From there it is transferred to CoA-SH forming acetyl CoA. The product is released. 5. E3 oxidizes lipoamide by transferring two H-atoms to the cofactor FAD. 6. FADH2 is oxidized by NAD+ and the enzyme complex is ready for the next cycle.  Malfunction of pyruvate dehydrogenase complex: TPP deficiency ⚫ Vitamin B1 (thiamine) deficiency leads to TPP deficiency that inhibits the enzymes requiring TPP as coenzyme. ⚫ Pyruvate dehydrogenase (E1) is inhibited. Pyruvate oxidation (=glucose oxidation) is inhibited. Brain suffers. ⚫ Dr. Jacob Bonitus, a Dutch physician working in Java, first described the disease Beriberi in 1630. ⚫ Symptoms: neurological and cardiovascular disorder. ⚫ Detection: increase in pyruvate in blood. Low enzyme activity (especially befurther transketolase) in blood. be they cannot ⚫ Treatment: supply vitamin B1 (unpolished rice). blood Coffee break  TCA cycle provides a way of cleaving a two-carbon compound The TCA cycle in the next slide appears to be a complicated way to oxidize acetate units to CO2 but there is a chemical basis for the apparent complexity. Oxidation of an acetyl group to a pair of CO2 molecules requires C-C cleavage: In many instances C-C cleavage reactions in biological systems occur between carbon atoms a and b to a carbonyl group: Another common type of C-C cleavage is a-cleavage of an a-hydroxyketone: Neither of these cleavage strategies is suitable for acetate. It has no b-carbon, and the second method would require hydroxylation – not a favorable reaction for acetate. Instead, living things have evolved the clever chemistry of condensing acetate with oxalacetate and then carrying out a b-cleavage. The TCA cycle combines this b-cleavages reaction with oxidation to form CO2, regenerate oxalacetate, and capture the liberated metabolic energy in NADH and ATP.  The concept of the TCA (Krebs) cycle ⚫ The citric acid cycle has eight steps, each catalyzed by a specific enzyme ⚫ The acetyl group of acetyl CoA joins the cycle by combining with oxaloacetate, forming citrate ⚫ The next seven steps decompose the citrate back to oxaloacetate, making the process a cycle ⚫ The NADH and FADH2 produced by the cycle relay electrons extracted from food to the electron transport chain CITRIC OXIDATIVE PYRUVATE GLYCOLYSIS ACID PHOSPHORYL- OXIDATION CYCLE ATION ATP © 2017 Pearson Education, Ltd  I to 5 : decarboxylat Acetyl CoA 6 to 8 : regenerate of oxaloacetate CoA-SH NADH + H+ H2O NAD+ Oxaloacetate 20] isomerise Malate Citrate Isocitrate CITRIC NAD+ NADH ACID Yo) + H+ hydrated H2O CYCLE CO2 Fumarate CoA-SH a-Ketoglutarate 50] CoA-SH FADH2 CO2 NAD+ FAD NADH Succinate Pi + H+ GTP GDP CoA ~ Succinyl this bond ADP high energy ATP © 2017 Pearson Education, Ltd  Formation of citrate by the Citrate synthase Acetyl CoA Acetyl CoA adds its two-carbon group to oxaloacetate, producing citrate; CoA-SH this is a highly exergonic reaction. H2O citrate (pdt) inhibits citrate Synthase Oxaloacetate Citrate ~ dehydrated can cleare carboxyl grp CITRIC hydrat Isocitrate ACID CYCLE ~ enzyme-bound 2 Step process cat by aconitase Citrate is a tertiary alcohol, meaning that the carbon atom bearing the hydroxyl group is bonded to three other carbon atoms. This presents yet another problem: Tertiary alcohols cannot be oxidized without breaking a carbon-carbon bond. This is because the carbon atom bearing the hydroxyl group, already bonded to the three other carbons, cannot from a carbon-oxygen bond. To set up the next oxidation in the pathway, citrate is first converted to isocitrate, a chiral secondary alcohol, which can be more readily oxidized. This isomerization reaction, catalyzed by aconitase, involves successive dehydration and hydration, through cis-aconitase intermediate, which remains enzyme-bound. © 2017 Pearson Education, Ltd D. R. Appling, S. Anthony-Cahill, C. K. Mathews, Biochemistry: Concepts and Connections, 2nd edition (2019)  Formation of Succinyl CoA Acetyl CoA CoA-SH H2O Oxaloacetate Citrate Isocitrate Redox reaction: Isocitrate is CITRIC NAD+ NADH oxidized; NAD+ is reduced. ACID + H+ CYCLE CO2 CO2 release CoA-SH a-Ketoglutarate CO2 CO2 release NAD+ NADH Redox reaction: After CO2 release, the + H+ resulting four-carbon molecule is oxidized energy Succinyl (reducing NAD+), then made reactive by CoA addition of CoA. © 2017 Pearson Education, Ltd high bond Mechanism of the NAD+-dependent isocitrate dehydrogenase Mechanism of the isocitrate dehydrogenase reaction - ɑ β Co) takes place 1st & beto a gup NAD+-dependent isocitrate dehydrogenase, is thought to catalyze the oxidation of a secondary alcohol (isocitrate) to a ketone (oxalsuccinate) followed by the decarboxylation of the carboxyl group to give the product, ɑ-ketoglutarate. in close proximity ~close to / In this sequence, the keto group b to the carboxyl group facilitates the decarboxylation by acting as an electron sink. The oxidation occurs with the reduction of NAD+. Mn2+ coordinates the formed carbonyl group so as to polarize its electronic charge. Voet, Voet: BIOCHEMISTRY 3rd edition  Mechanism of the NAD+-dependent a-Ketoglutarate Dehydrogenase TPP involved Mechanism of the a-Ketoglutarate Dehydrogenase reaction α-ketoglutarate dehydrogenase catalyzes the oxidative decarboxylation of an α-keto acid (α- ketoglutarate), releasing the citrate acid cycle’s second CO2 and NADH. The overall reaction, which resembles that catalyzed by the PDC (see slide 13), is mediated by a homologues multienzyme complex consisting of α-ketoglutarate dehydrogenase (E1), dihydrolipoyl transsuccinylase (E2) and dihydrolipoyl dehydrogenase (E3). NADH and Succinyl-CoA, produced in this catalytic steps, are energy rich and important sources of metabolic energy. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Mechanism of the Succinyl-CoA Synthetase Acetyl CoA CoA-SH The NADH produced in the foregoing steps can be routed through the electron transport H2O pathway to make high-energy phosphates via oxidative phos- Oxaloacetate phorylation. Succinyl-CoA is itself a high-energy intermediate Citrate and is utilized in the next step of Isocitrate the TCA cycle to drive the CITRIC NAD+ phosphorylation (substrate- NADH level phosphorylation) of GDP ACID + H+ to GTP (in mammals) or ADP to CYCLE CO2 ATP (in plants and bacteria). CoA-SH a-Ketoglutarate CoA-SH CO2 NAD+ NADH Succinate Pi high + H+ GTP GDP Succinyl CoA ADP bond energy ATP-formation ATP © 2017 Pearson Education, Ltd  Reactions catalyzed by succinyl-CoA synthetase Step 1: Formation of succinyl phosphate, a “high-energy” mixed anhydride imidazole viny undergo we attack int Histidine Step 2: Formation of phosphoryl–His, a “high-energy” intermediate residue within - enzyme succinyl-loA synthetase Histidine regenerated Step 3: Transfer of the phosphoryl group to GDP, forming GTP Reactions catalyzed by the succinyl-CoA synthetase Step 3: Transfer of the phosphoryl group to GDP, forming GTP The GTP produced by mammals in this reaction can exchange its terminal phosphoryl group with ADP via the nucleoside diphophate kinase reaction: Nucleoside diphosphate kinase GTP + ADP ATP + GDP reversible  Reaction catalyzed by the Succinate Dehydrogenase Succinate Dehydrogenase has FAD covalently Acetyl CoA bound. FAD oxidizes alkanes to alkenes, whereas CoA-SH NAD+ oxidizes alcohols to aldehydes or ketones. This is because the oxidation of an alkane is sufficiently exergonic to reduce FAD to FADH2, but not to reduce H2O NAD+ to NADH. Being linked to the succinate dehydrogenase (also called Complex II) FADH2 Oxaloacetate becomes reoxidized by coenzyme Q in the electron transport chain. Succinate Dehydrogenase is the only membrane-bound TCA cycle enzyme. Citrate Isocitrate CITRIC NAD+ NADH ACID + H+ CYCLE CO2 eving is Fumarate CoA-SH fixed to histidine a-Ketoglutarate To) in succinate CoA-SH dehydrogenase FADH2 CO2 NAD+ FAD Redox reaction: NADH & N, take Succinate Pi Ns can over Succinate is oxidized; GTP GDP Succinyl + H+ 2e- & 2 protons FAD is reduced. CoA ADP ATP © 2017 Pearson Education, Ltd  Acetyl CoA CoA-SH NADH hydrat + H+ H2O NAD+ Oxaloacetate [0) Malate Citrate Isocitrate CITRIC NAD+ NADH hydrat ACID + H+ H2O CYCLE CO2 Fumarate CoA-SH a-Ketoglutarate CoA-SH FADH2 CO2 NAD+ FAD NADH Succinate Pi GTP GDP Succinyl + H+ + 3 MADH CoA ADP + 1 FADHL ATP © 2017 Pearson Education, Ltd + 1 ATP/Gyp  The fate of carbon in the TCA cycle C coming from acetyl cont are note carbons that are released by co2 (from oxaloacetate) The oxidation–reduction enzymes and coenzymes are shown in magenta. Entry of the two carbons of acetyl-CoA into the TCA cycle are indicated with the green box. The carbons released as CO2 are shown with yellow boxes. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  What are the energetic consequences of the TCA cycle in mammals? ⚫ Acetyl-CoA + 3 NAD+ + FAD + ADP + Pi + 2 H2O → 2 CO2 + 3 NADH + 3 H+ + FADH2 + CoASH + ATP ⚫ Glucose metabolized via glycolysis produces two molecules of pyruvate and thus two molecules of acetyl-CoA, which can enter the TCA cycle. Combining glycolysis and the TCA cycles gives net reaction shown: Glucose + 10 NAD+ + 2 FAD + 4 ADP + 4 Pi + 2 H2O → 6 CO2 + 10 NADH + 10 H+ + 2 FADH2 + 4 ATP Acetate is completely oxidized to CO2, and the free energy is conserved. by e carriers Note that in this complete oxidation no free O2 molecule is involved. Quiz 1: During one cycle, 2 carbon atoms are oxidized to CO2. Where do these carbon atoms come from? Voet, Voet: BIOCHEMISTRY 3rd edition  Standard Free Energy Changes (DG°) and Physiological Free Energy Changes (DG) of Citric Acid Cycle Reactions these are is enzymes that are regulated in TCA cycle The values show that three of the enzymes are likely to function far from equilibrium under physiological conditions (negative DG). Voet, Voet: BIOCHEMISTRY 3rd edition  Major regulatory interactions in the TCA cycle The rate of ATP hydrolysis controls the rate of ATP synthesis, which controls the rate of NADH oxidation in the electron-transport chain (ETC). All NADH and FAD(2H) produced by the cycle donate electrons to this chain. Thus, oxidation of acetyl-CoA in the TCA cycle can go only as fast as electrons from NADH enter the electron- I transport chain, which is controlled by the ATP inhibit and ADP content of the cells. The ADP and NADH concentrations feed information on the rate of oxidative phosphorylation back to the TCA cycle. Isocitrate dehydrogenase (DH), α- ketoglutarate DH, and malate DH are inhibited by increased NADH concentration. The NADH/NAD+ ratio changes the concentration of oxaloacetate. Citrate is a product inhibitor of citrate synthase. ADP is an allosteric activator of isocitrate DH. During muscular contraction, increased Ca2+ concentrations activate isocitrate DH and α- ketoglutarate DH (as well as pyruvate DH). C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  Factors controlling the activity of the PDC L pyruvate dehydrogenase complex NADH and acetyl-CoA, respectively, compete with NAD+ and CoA in Reaction 3 and 5 of the PDC reaction sequence. inhibits  Anaplerotic pathways to replenish TCA cycle intermediates Major anaplerotic pathways of the refilling tricarboxylic acid cycle. (1) and (3) (red arrows) oxalostate are the two major anaplerotic pathways. (1) Pyruvate carboxylase. transaminat (2) Glutamate is reversibly converted to α- forms X2 ketoglutarate by transaminases (TA) and glutamate dehydrogenase (GDH) in many tissues. (3) The carbon skeletons of valine and isoleucine, a three-carbon unit from odd-chain fatty acid oxidation, and a number of other compounds enter the TCA cycle at the level of succinyl-CoA. Other amino acids are also degraded to fumarate (4) and oxaloacetate (5) principally in the liver. C. M. Smith, A. D. Marks & M. A. Liebermann, Marks’ Basic Medical Biochemistry: A Clinical Approach, 7th edition  The TCA cycle provides intermediates for biosynthetic processes TCA also oppositely , cycle generates precursor molecules for other pathways e 7 F A... -.. decarboxylated Garett & Grisham: Biochemistry 4th edition  Quiz 1. What is the DGo’ value for hydrolysis of the thioester bond of Acetyl-CoA? DGo’ = -31.5 KJ/mol than Hip hydrolysis slightly higher 2. What is the functional group in the thiazolium ring of TPP? & acidic proton C2 of thiazolium ring  Quiz NADH (reduced MAD ) + ~ 3. How can the reduction of NAD+ spectroscopically be observed? peak of reduced MAD + (MAPH) e 340 um observed UV-spectrum of NAD+ () and NADH (○), respectively  Quiz 4. Why is the Krebs cycle called Tricarboxyl Acid cycle (TCA)? 3 carboxylic - acid in citrate - & isocitrate  Quiz 5. Which of the 8 enzymes of the TCA cycle is membrane bound and which cofactor does it use in the catalytic reaction? complex 11 -membracee cofactor FAD is  Quiz 6. Name the enzymes of the TCA cycle, which are likely to function far from equilibrium under physiological conditions (negative DG)? 3 enzymes requisited in TCA © 2017 Pearson Education, Ltd. Thank you!