hapter 17 - itric Acid ycle I. Introduction - The citric acid cycle (A) was elucidated in the 1930's by ans Krebs, who first noticed that oxygen consumption in suspensions of pigeon breast muscle was greatly enhanced by addition of various di- and tri-carboxylic acids (which turned out to be A intermediates). - The reactions of the A, as well as those of various other oxygen-requiring pathways, such as oxidative phosphorylation and fatty acid catabolism, occur in the mitochondria, organelles which specialize in such aerobic processes. - Pyruvate is the end point of glycolysis, which occurs in the cytoplasm. Pyruvate must enter the mitochondria, where it is converted into acetyl oa, a high-energy form of acetate, which we encountered in hapter 14: 3 oa 3 NAD NAD The thioester (sulfur) linkage is the source of high energy in this molecule. It is destabilized relative to its hydrolysis products because the sulfur atom is so much larger than the carbon atom that sideways overlap of p orbitals to form the double bond is unfavorable. - This is a very complicated reaction, involving three distinct enzymes, several copies of which form a multi-million dalton enzyme complex, the pyruvate dehydrogenase complex. This efficient evolutionary development allows pyruvate to be processed, via an assembly-line fashion, into acetyl oa. This enzyme complex requires several cofactors, including NAD +, because this is an oxidation, and oenzyme A (to activate the acetate group). The only other coenzyme we need be concerned with is thiamine pyrophosphate (TPP - thiamine is the vitamin precursor), which helps catalyze the carbon-carbon cleavage, which occurs between the carbonyl carbon and the "-carbon (an "- cleavage). TPP is attached to the substrate, temporarily changing the molecular structure of pyruvate, giving rise to a $-cleavage, as occurs in glycolysis: ' ' N 3 ' ' N 3 oa ' N 3 TPP Pyruvate E complex NAD + NAD TPP Acetyl oa oa - The oa moiety of acetyl oa serves only to activate the 2-carbon acetyl group prior to entering the A. These must be cleaved, but neither an "- nor $-cleavage is possible with so few carbons. Nature has therefore devised a cyclic, catalytic, pathway to achieve this goal. Note that the A is cyclic in nature because a catalyst must be regenerated. Nature has thus devised a cyclic, rather than a linear pathway like glycolysis, to regenerate the catalyst:
oa 3 atalyst 2 2 II. Individual reactions of the A Acetyl oa oa 3 3 2 xaloacetate 2 2 itrate 2 Isocitrate 2 Malate 2 Fumarate uccinate 2 2 alpha-ketoglutarate 2 uccinyl oa 2 2 2 2 oa 2 1. The activated acetate is attached to the catalyst, oxaloacetate to form citrate, an energyrequiring process which is driven to completion by the hydrolysis of the high-energy thioester linkage. 2. itrate has no carbonyl group, which is essential for the carbon-carbon cleavages which
must occur to produce carbon dioxide. itrate is thus isomerized to isocitrate to reposition a hydroxyl group so it can be oxidized to a carbonyl (see top of page 475). Note that this reaction is not shown in figure 17.15). 3. The above-mentioned hydroxyl is oxidized to a carbonyl, resulting in a spontaneous $- cleavage to form "-ketoglutarate ("kg, see p. 475 and Figure 17.15) 4. "kg is structurally very similar to pyruvate and is oxidatively cleaved by a similar mechanism as the pyruvate dehydrogenase-catalyzed reaction to form succinyl oa and producing the second 2. The catalytic goal has now been accomplished. 5. Whereas the energy inherent in the thioester linkage in acetyl oa was used to form citrate, in this case the energy will be harvested to form ATP in a substrate level phosphorylation, yielding succinate as a product. 6-8. The last three steps in the A regenerate the original catalyst, oxaloacetate: An oxygen is required at -2, so succinate is oxidized to fumarate, which is hydrated to form malate, which is then oxidized to form oxaloacetate: FAD FAD 2 NAD + NAD III. The citric acid cycle as a source of metabolites for other pathways. - everal pathways intersect the A, with common intermediates at the intersection points. These include "-ketoglutarate (amino acids and nitrogen metabolism), oxaloacetate (amino acid metabolism, gluconeogenesis), and succinyl oa (porphyrin synthesis). Due to the catalytic nature of the cycle, A intermediates are not plentiful and must be replenished if siphoned off into other pathways. ence the following, anaplerotic reaction ( to fill up ): biotin 3 ATP ADP 2 Pyruvate oxaloacetate IV. Glyoxylate cycle - Allows seeds and plants to grow on fats by altering the A (see next page). V. egulation - The pyruvate dehydrogenase reaction commits pyruvate to either the A or fatty acid, then lipid metabolism. ence this reaction must be regulated. - ontrolled reactions within the cycle are the entry point or acetyl oa and the two decarboxylations:
Pyruvate ATP, Acetyl oa, NAD Acetyl oa ATP itrate xaloacetate Isocitrate Malate ATP ADP Alpha-ketoglutarate Fumarate uccinyl oa uccinyl oa NAD uccinate IV. Glyoxalate cycle continued: Note that reactions 1-7 result in the conversion of acetyl oa to glucose. nly reactions 3 and 4 are unique to the glyoxalate cycle; the others are involved in either the A or gluconeogenesis (reactions 6 and 7 (7 is actually 10 reactions comprising gluconeogenesis)). eactions 8, 9 and 10 of the A are required to convert succinate, one of the cleavage products of reaction3, back to oxaloacetate to regenerate the original catalyst. VI. Problems: 1, 2, 5, 6 VII. You re responsible for the following pages: 465-467, 472-478, 480 -end. You need know the mechanism of the pyruvate dehydrogenase complex only as discusses in class (section 17.1)
3 4 9 3 2 2 oa 2 2 2 2 3 oa 2 2 2 2 2 2 2 2 3 oa fatty acids Glucose 1 2 5 6 7 10