Biochemistry ; Lecture TCA & Glyoxylate Cycles There is no reason for this except I love Paris! What flows from the TCA cycle to ETS is the NADH. It is the oxidation of NADH drives the production of ATP. The TCA intermediates function in other pathways, the product of this oxidative pathway, NADH is the substrate for the ETS. THE starting substrate for the TCA cycle is acetylCoA, that can be derived from; 1.pyruvate or 2. fatty acids through - oxidation. Why is this called an amphibolic pathway? Is this only found in aerobes? In aerobes it is an oxidative pathway, in anaerobes is it a reductive pathway? 3NAD + + FAD + +GDP + P i + Acetyl-CoA 3NADH + FADH 2 + GTP + CoA + 2CO 2 Electron micrographs of the E. coli pyruvate dh multienzyme complex, approx 300 angrstroms. Range of size is 300 to 500 A. Stucturally as well as mechanistically there is not difference between the prokaryotic Pdh & the eukaryotic Pdh. The difference is in the number of peptides of each specific peptide in the protein. The Pdh is a model for Keto acid dh, such as KGdh is an identical structure but E1 has a different specificity in each keto acid dh. The product of this pathway is a highly reactive substrate for the TCA cycle, acetyl-CoA. t will condense with OAA to form citrate. The rate limiting substrate for this pathway is NAD +. It is reduced by the enzyme lipoamide dh, and then feeds its H + (NADH is oxidized) into the ETS to generate ATP. 4H + produced reduces O 2 molecule. This pathway is a multi-enzyme complex that functions as a structure. The relationship of E1, E2 & E3 and how lipoamide acts as a structural extension of the transacetylase. Lipoamide forms a 14A arm that binds the acetyl group that is transferred from TPP in the the E1 active site. Lpd brings the acetyl grp to the E2 active site where the acetyl group is transferred to CoA. It is a transfer from the thiol of lipoamide to the thiol of CoA. Structure of E 1 from P. putida branched-chain a-keto acid dehydrogenase. An a 2 b 2 heterotetrameric protein. Structure of dihydrolipoamide dehydrogenase (E3) from P. putida in complex with FAD + and NAD +. A homodimeric enzyme. Structure of E1 from P. putida branched-chain a-keto acid dehydrogenase. A surface diagram of the active site region. Structure of dihydrolipoamide dehydrogenase (E3) from P. putida in complex with FAD + and NAD +. The enzymes active site region. Cys 43 & 48 are found on an destorted -helixes and a deep FAD + binding site with NAD + very close. The phenolic group of Tyr 181 protects the e - transfer. FAD + has a reduction potential of V0, and NAD + o of V. Structure of a trimer of A. vinelandii dihydrolipoyl transacetylase (E2) catalytic domains. Lipoamide is assoc with Lys residue on E2 protein. Factors controlling the activity of the Pdh product inhibition. Factors controlling the activity of the Pdh. Covalent modification in the eukaryotic complex. 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 3 moles of NADH and 1 mole of FADH2 generated during each round of the cycle feed into the oxidative phosphorylation pathway. Each mole of NADH leads to 3 moles of ATP and each mole of FADH 2 leads to 2 moles of ATP. Therefore, for each mole of pyruvate which enters the TCA cycle, 12 moles of ATP can be generated. The overall stoichiometry of the TCA cycle is: acetyl-CoA + 3NAD + + FAD + + GDP + P i + 2H 2 O ----> 2CO 2 + 3NADH + FADH 2 + GTP + 2H + + HSCoA Note the endothermic Mdh, +29.7kJ/mol and the exothermic citrate synthase, kJ/mol. Why should Mdh have evolve to favor malate and not OAA? Regulation of the citric acid cycle. Standard Free Energy changes (G) and physiological Free Energy changes (G) of citric acid cycle reactions. Structure of citrate synthase. The flexible domain of each subunit undergoes a large conformational change on binding OAA, creating a binding site for acetyl-CoA. (a) Open form of the enzyme alone; (b) closed form with bound OAA and a stable analog of acetyl-CoA. In these representations one subunit is colored tan and one green. MECHANISM; Citrate synthase. In the mammalian citrate synthase rxn,OAA binds first, in a strictly ordered reaction sequence. This binding triggers a conformation change that opens up the binding site for acetyl-CoA. OAA is specifically oriented in the active site of citrate synthase by interaction of its two carboxylates with two positively charged Arg residues (not shown here). Iron-sulfur center in aconitase. The iron-sulfur center is in red, the citrate molecule in blue. Three Cys residues of the enzyme bind three iron atoms; the fourth iron is bound to one of the carboxyl groups of citrate and also interacts non-covalently with a hydroxyl group of citrate (dashed bond). A basic residue (:B) in the enzyme helps to position the citrate in the active site. The iron-sulfur center acts in both substrate binding and catalysis. This reaction is to convert the tertiary alc to a an easily oxidized secondary alc, isocitrate. Isocitrate dehydrogenase. In this reaction, the substrate, isocitrate, loses one carbon by oxidative decarboxylation. This occurs with the oxidation of isocitrate to oxalosuccinate Reducing NAD + to NADH, then the decarboxylation of oxalosucc to -KG. This is another member of the Keto acid dh enzymes like Pdh. The E1 is the KG dh. Succinyl-CoA synthetase reaction. 1 a phosphoryl group replaces the CoA of succinyl-CoA bound to the enzyme, forming a high-energy acyl phosphate. 2 the succinyl phosphate donates its phosphoryl group to a His residue of the enzyme, forming a high-energy phosphohistidyl enzyme. 3 the phosphoryl group is transferred from the His residue to the terminal phosphate of GDP (or ADP), forming GTP (or ATP). The succinyl-CoA synthetase reaction. Active site of succinyl-CoA synthetase of E. coli. The active site includes part of both the (blue) and the (brown) subunits. The power helices (blue, brown) place the partial positive charges of the helix dipole near the phosphate group of PHis 246 in the chain, stabilizing the phosphohistidyl enzyme. The bacterial and mammalian enzymes have similar amino acid sequences and three- dimensional structures. In the reverse rxn this is fumerate reductase. Facultative aerobes Like E.coli have both a succinate dh & fumerate reductase gene, but structually they are identical. In the mitochondria this is a trans-membrane bound protein that is part of electron transport, complex II. Fumarase (or fumarate hydratase) is an enzyme that catalyzes the reversible hydration/dehydration of fumarate to malate. Fumarase comes in two forms: mitochondrial and cytosolic. The mitochondrial isoenzyme is involved in the Krebs, and the cytosolic isoenzyme is involved in the metabolism of amino acids and fumarate. Subcellular localization is established by the presence of a signal sequence on the amino terminus in the mitochondrial form, while subcellular localization in the cytosolic form is established by the absence of the signal sequence found in the mitochondrial variety 3NAD + FAD +GDP + P i + Acetyl-CoA 3NADH + FADH 2 + GTP + CoA + 2CO 2 The NADH and FADH formed are the substrates for the ETS and OX-OHOS. What is the reason for this shuttle pathway? How does this pathway give the cell an advantage? The amino acids may be grouped for discussion on the basis of the specific keto acid products of their deamination. The 3-carbon a-keto acid pyruvate is produced from alanine, cysteine, glycine, serine, & threonine. Alanine deamination via Transaminase directly yields pyruvate The 4-carbon Krebs Cycle intermediate oxaloacetate is produced from aspartate & asparagine. Aspartate deamination via transaminase directly yields oxaloacetate. Aspartate also is converted to fumarate in the Urea Cycle. Fumarate is then converted by Krebs Cycle enzymes in the cytosol (fumerase) to malate, and by Mdh to oxaloacetate. The 4-carbon Krebs Cycle intermediate succinyl-CoA is produced from isoleucine, valine, & methionine. Propionyl-CoA, which is an intermediate on these pathways, is also a product of -oxidation of fatty acids with an odd number of C atoms fatty acid chains. The 5-carbon Krebs Cycle intermediate a-ketoglutarate is produced from arginine, glutamate, glutamine, histidine, and proline. Glutamate deamination, via glutamate dehydrogenase or transaminase, directly yields -ketoglutarate The Urea cycle which is the cornerstone of Nitrogen metabolism in mammalian cells, is closely tied to the TCA Cylce. Glyoxylate cycle What makes this pathway unique is that it produces succinate & glyoxylate from Isocitrate. It is an incomplete TCA cycle that has two unique enzymes? What are they? What advantage does this pathway give to a cell? Isocitrate lyase is an enzyme that functions at a branch point of carbon metabolism and diverts isocitrate through a carbon- conserving pathway, the glyoxylate cycle, bypassing the two decarboxylative steps of the tricarboxylic acid cycle that convert isocitrate to succinyl- CoA. The glyoxylate molecule formed by isocitrate lyase reaction condenses with one acetyl-CoA to produce L-malate in the subsequent step catalyzed by malate synthase (the other enzyme unique to the glyoxylate cycle). acetyl-CoA + glyoxylate + H 2 O = L-malate + CoA In what organisms would this sort of TCA cycle function? It appears that the two main products of this pathway are succinyl-CoA and KG. IN a reductive pathway what are the possible end products? PNAS-2004-Smith