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CHAPTER 16 - CITRIC ACID CYCLE Introduction: - Glycolysis and other catabolic pathways are oxidative. Electrons are stripped from a fuel substrate (e.g., glucose) and passed to the appropriate electron acceptor, which thus is reduced. Recall that in glycolysis, pyruvate was reduced to lactate (which is the same oxidation state as glucose) under anaerobic conditions because need to rapidly regenerate NAD+, which had received electrons as glyceraldehyde 3-phosphate was oxidized to 1,3-bisphosphoglycerate. - Normally, in our cells, and in other aerobic organisms, electrons are passed to O2 as fuel molecules are oxidized. When we are not exercising rigorously, pyruvate, the end product of glycolysis, passes into the mitochondria, where it is oxidatively decarboxylated to acetyl CoA which then enters the citric acid. As we will see in chapter 17, the reduced cofactors NADH and FADH2 then pass their electrons through a membrane-bound electron transport chain to the ultimate electron acceptor, O2. Such oxygen-requiring processes are termed (cellular) respiration. - Several other fuels, including proteins (6 amino acids) and fats funnel metabolites into the citric acid cycle, just as glycolysis funnels in pyruvate. - The citric acid cycle also functions to provide reactants for a variety of biosynthetic pathways. - Historically, the citric acid puzzle was solved in the ‘30's, although the final picture didn’t come together until the early ‘50's when acetyl CoA was shown to be the catabolic intermediate that brings pyruvate into the cycle. - As glucose is broken down into smaller products, it becomes increasingly difficult to break bonds. Acetyl CoA is essentially a 2-carbon fragment of glucose and cannot undergo either alpha or beta cleavages. The citric acid cycle is a catalytic pathway whose purpose is to facilitate the breakdown of acetate into CO2 . Acetyl CoA condenses with a catalyst (oxaloacetate), thus producing a larger molecule (citrate) which can then undergo structural rearrangements that result in first a beta, then an alpha cleavage. The catalytic process is cyclic because of the need to regenerate the catalyst. Pyruvate Acetyl CoA Citrate catalyst beta CO2 Regenerate catalyst alpha CO2 - The catalytic nature of the citric acid cycle was originally suggested by the finding that addition of small amounts of various dicarboxylic and tricarboxylic acids greatly accelerated the rate of oxygen consumption in muscle tissue, beyond expectations based on the oxidative capacity of the acid itself. Thus, these substances were catalyzing the oxidation of other foodstuffs. - There are several oxidations which occur during the pyruvate 6 CO2 conversion in mitochondria. There are three requiring NADH as cofactor and 1 requiring FAD. As we’ll see in the next chapter, electrons from the reduced versions of these cofactors (NADH and FADH2) feed their electrons into the electron chain, ultimately to O2. The electron transport process is energy-yielding and is coupled to ATP formation (oxidative phosphorylation). The majority of the 36 to 38 ATP extracted per glucose under aerobic conditions is via oxidative phosphorylations (3 per NADH and 2 per FADH2). Synthesis of Acetyl CoA - It is useful to compare the aerobic conversion of pyruvate to acetyl CoA to the anaerobic conversion of pyruvate to acetaldehyde in yeast: O O C C yeast H CH3 mitochondria S CoA O C O C CH3 CH3 - Both of these reactions involve alpha decarboxylations, hence require thiamine as a cofactor. See Figure 14-20 for a depiction of the thiamine-catalyzed alpha decarboxylation. - Formation of acetyl CoA is a much more complicated process than the formation of acetaldehyde and in eucaryotes is catalyzed by a multi-million molecular weight complex, the pyruvate dehydrogenase complex, consisting of multiple copies of three enzymes, E1, E2 and E3 (E1 = pyruvate dehydrogenase; E2 = dihydrolipoyl transacetylase; E3 = dihydrolipoyl dehydrogenase). - E2 is the core of the complex and covalently binds lipoate via an amide linkage to a lysine residue: HS HS NH CH2 CH2 CH2 CH2 CH2 CH2 CH2 C E2 O Lipoate Lysine - The methylene groups of the lipoate together with the lysine R group forms a long “tether” group that swings the sulfur groups, to which substrate binds, from E1 to E2 to E3. R 1 N C H S R 2 R3 O + O C Mech C O anism: CH3 Let’s pick E1 up the R 2 R action R O O 1 C N C C OH S CH3 3 with E1, which O C O R 1 N C S R 2 R H bound CH3 thiamin e: H+ CH3 R3 C OH 3 C R 2 sa O + R 1 N C H S contain + + H+ R 1 N R 2 C S R 3 (yeast only) S C OH S E2 Tether - CH3 (pyruvate dehydrogenase only R 2 R 3 HS R 1 N S C S C OH CH3 E2 Tether - HS R 1 N R 2 S C Tether - C OH S R 3 E2 CH3 B + H+ R 2 R 3 R 1 N CH S HS E2 S + C O Tether CH3 - At this point the pyruvate has undergone oxidative decarboxylation. A thioester linkage has been formed between substrate and lipoate. An internal transfer of electrons has occurred such that the original C2 of pyruvate has been oxidized to the oxidation state of a carboxylate group (S “owns” the electrons in the C-S bond) and the lipoate has been partially reduced. - Note that the lipoate group is capable of existing in either a completely reduced form or oxidized form: HS HS E2 Tether - -H + H + + H + H+ S S E2 Tether - - E2 must now catalyze the transfer of the acetyl moiety from the lipoate to a CoA(SH) molecule and the reduced lipoate must transfer a pair of electrons to an external electron acceptor (NAD+): HS E2 S C O Tether CH3 CoASH HS E2 HS SCoA C O Tether CH3 FAD (E2 - bound) S E2 S FADH2 Tether FAD This reaction can be summarized as follows: CoA Acetyl CoA Pyruvate Lipoate Lipoate (oxidized) (reduced) FADH2 FAD NAD + NADH NAD+ NADH O C S CoA CH3 CO2 CH2 1 NADH + NAD CO2 HO CCO2 CH2 CO2 C O C O2 CH2 CO2 CH2 9 2 HC CO2 HO CH CO2 NAD+ C O2 HO CH NADH 3 CH2 CO2 CO2 CH2 HCCO2 O C 8 CO2 C O2 CH HC CO2 CH2 C O2 7 CH2 CO2 FADH2 6 CH2 CO2 O C C O2 CH2 FAD 4 CH2 CH2 GTP O C GDP S CoA CO2 5 NAD+ NADH Notes: 1. Acetyl CoA condenses with the catalyst, oxaloacetate, to enter the citric acid cycle. Citrate, the product, has 6 carbons which can now be arranged to facilitate decarboxylations by either alpha or beta cleavages. 2. Citrate is isomerized to isocitrate because the hydroxyl group on citrate is on a tertiary carbon and cannot be oxidized to a carbonyl. Recall that the OH group on isocitrate is now on the portion of the molecule originally derived from oxaloacetate. See Figure 20-15 to refresh your memory as to why the OH never gets bound to a carbon originally from acetyl CoA. 3. Isocitrate is oxidized to oxalosuccinate, an unstable intermediate which spontaneously undergoes beta decarboxylation to form alpha ketoglutarate (step 4.). 5. Alpha keto glutarate undergoes alpha decarboxylation to form succinyl CoA in a manner uncannily similar to that of the pyruvate to acetyl CoA reaction considered in detail above. 6. Whereas the high-energy acetyl CoA was put to use to drive the otherwise energetically unfavorable formation of citrate, the high-energy succinyl CoA is hydrolyzed to drive the formation of GTP, our third (and last) substrate level phosphorylation. 7. - 9. The last three reactions regenerate the catalyst by successively forming a double bond, adding water across the double bond and oxidizing the hydroxyl group to a carbonyl, yielding oxaloacetate. Other notes: - Because the citric acid cycle is an important source of precursors for other pathways (citrate, oxaloacetate, succinyl CoA and alpha keto glutarate can be drawn off - see Figure 20-17), the concentrations of citric acid cycle intermediates can fall to unacceptably low levels. Recall that because of the catalytic nature of the cycle, these levels are fairly low to begin with. To prevent this from happening, pyruvate can be converted to oxaloacetate (anaplerotic reaction). We recognize this as the first step in gluconeogenesis: C O2 CO2 C O2 biotin C O CH3 C O CH2 ATP ADP CO2 Amphibolic Nature of cycle - In addition to catalyzing the conversion of acetyl CoA into CO2, the citric acid cycle also ser ves Pyruvate Fatty acids to Cholesterol pro vid e Amino Acids Acetyl CoA Citrate Oxaloacetate inte Isocitrate rme diat Malate es Alpha ketoglutarate for sev Amino Acids Fumarate Succinyl CoA eral Amino oth Acids er pathways. Succinate Odd- chain fatty acids Amino Porphyrins Acids - Because of the catalytic nature of the cycle, its intermediates are present in low, catalytic amounts. Should the drain of these intermediates into any of these pathways, citric acid cycle activity could be compromised, which is unacceptable. The following anaplerotic (“filling up”) reaction is one of a few that replenish citric acid cycle intermediates: O O O C O C C O C O CH2 CH3 Pyruvate ATP ADP C O O Oxaloacetate - You will recognize this reaction as the first step in gluconeogenesis. Be sure to make note of this in your metabolic road map. Regulation - The four NADH, 1 FADH2 and 1 GTP obtained from converting pyruvate to CO2 results in 4 x 3 + 1 x 2 + 1 = 15 ATP per pyruvate, or 30 per glucose. Such a large amount of ATP necessitates tight regulation of pyruvate to acetyl CoA as well as the citric acid cycle itself. - Regulation of the pyruvate dehydrogenase complex occurs mechanistically by product inhibition and covalent modification. NADH and acetyl CoA as products thus compete for binding sites on E2 and E3. Since NADH is also an electron donor, large amounts of NADH (or large [NADH][/NAD+] ratios) can reverse the flow of electrons, ultimately preventing the oxidative decarboxylation of pyruvate by E1. - E1 is also subject to covalent modification by a regulatory kinase (which is allosterically activated by the high-energy indicator, ATP). Phosphorylation of E1 results in its inactivation. Note that insulin, which we have previously seen to have antagonistic effects with respect to glucagon and epinephrine, and which terminates glycogen breakdown and initiates glycogen synthesis, reverses the inactivation of E1 by activating the phosphoprotein phosphatase which removes the phosphate group on E1. This is consistent with insulin’s role in accelerating glucose uptake as well as stimulating cellular processes that use up or store glucose. - Regulation of the cycle itself occurs at the three exergonic reactions, i.e., the synthesis of citrate (involving the hydrolysis of the high-energy thioester bond in acetyl CoA) and the two decarboxylations. Although allosteric modulators are involved in regulation of the cycle (as high-energy indicators, they have an inhibitory effect), the primary mechanisms are substrate availability(1), product inhibition(2) and competitive feedback(3). Pyruvate NADH (2) * Acetyl CoA(1) NADH (2) * Citrate Oxaloacetate(1) Isocitrate * NADH (2) Malate Alpha ketoglutarate * NADH (2) Fumarate Succinyl CoA (3) Succinate - Note that the (3) next to succinyl CoA implies that succinyl CoA inhibits via competitive feedback inhibition. In this case the reaction inhibited in the citrate synthase reaction, which is also inhibited by acetyl CoA. See Figure 16-14, p. 486. Glyoxalate Cycle - Seeds store fats as both a source of energy and precursors for carbohydrate. The glyoxalate cycle is properly thought of as a variation of the citric acid cycle. O C S CoA CH3 CO2 CH2 NADH + NAD HO CCO2 CH2 CO2 C O C O2 CH2 CO2 CH2 HC CO2 HO CH CO2 CO2 C O2 O HO CH CH2 C CO2 CH3 NAD+ NADH S CoA H O CO2 1 CH2 C 2 HCCO2 O C C O2 CO2 C O2 CH HC CO2 CH2 C O2 CH2 CO2 FADH2 FAD CH2 C O2 CH2 CH2 CO2 O C CO2 CH2 GTP O C GDP S CoA NAD+ NADH - The glyoxalate cycle includes citric acid cycle reactions up to the formation of isocitrate. Isocitrate is then cleaved to form glyoxalate and succinate in the first reaction unique to seeds (reaction #1). Then glyoxalate is added to a second acetyl CoA in reaction #2 to form citrate. - In this way, via the actions of two additional enzymes, isocitrate lyase and malate synthase, seeds avoid the decarboxylation steps of the citric acid cycle. The catalyst can be regenerated from succinate, one product of the lyase reaction, so the malate synthesized from the two acetyl CoA can be converted to oxaloacetate, then glucose via gluconeogenesis. See Figure to see how the reactions are compartmentalized between the various organelles.