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1、(also Tricarboxylic Acid Cycle, or Krebs cycle)The final common pathway involved in the oxidation of carbohydrates, fats, and proteins into CO2 and H2O to generate ATP by linking with oxidative phosphorylation); It also provides precursors for the biosynthesis of many compounds (e.g. amino acids); O
2、2 is needed only indirectly to regenerate NAD+ and FAD. Occurring inthe matrix ofmitochondriain eukaryoteslThe chemical reactions and enzymes of the citric acid cycle and the glyoxylate bypass (cycle, shunt).lThe elucidation of the citric acid cycle and the glyoxylate bypass.lSummaryC2C4 (Regenerate
3、d )C6 (tricarboxylic acid )C5C4This cycle neither generates a large amount of ATP nor include O2 as a reactant.Pyruvate dehydrogenasecomplexIn yeast cytoplasmIn mitochondrial matrixThe lipoamideVitamin B5Cryoelectron micrograph (PDH from bovine kidney)ReconstructedimageZ Hong Zhou2001, PNAS 98:14802
4、-7. E2DescriptionReactantsProductsAll reactions in the citric acid cycle:Acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2 H2O CoA-SH + 3NADH + 3 H+ + FADH2 + GTP + 2 CO2Further oxidation of each NADH provide enough energy for producing about 2.5 ATP molecules and each FADH about 1.5. Aldol condensationIron-s
5、ulfur protein Dehydration- rehydrationOxidative decarboxylationHydrolysisOxidationHydrationDehydrogenationlNo such phosphorylated enzyme intermediates formed in the two ATP formation steps in glycolysis!lTemporary complexes formed between sequential enzymes of a metabolic pathway.lAllowing passing (
6、substrate channelling) intermediary metabolic product from one active site to another of consecutive enzymes in the pathway.lDirect evidence still lacking. Boris I KurganovThe citric acid cycle also provides precursors for biosynthesis, and is thus amphibolic.Oxaloacetate isreplenished frompyruvate
7、and PEPThe carboxylation of pyruvate needs the participation of biotin (covalently linked to pyruvate carboxylase and switches between two active sites)(biotin binds to avidin with a Kd of 10-15 M)a a-ketoglutarate dehydrogenaseis not present in such organisms!An anabolic pathway:Converting acetyl-C
8、oA to glucose and other biomolecules,occurring in plants and certainmicroorganisms.Phosphoenoypyruvatemight be considered as the end product of theglyoxylate bypass.The partition of carbon fluxbetween the glyoxylate bypassand the citric acid cycle hasbeen under extensive studies.Reversible phosphory
9、lation (in E. coli);Km value differences;Malate synthase (1956)Isocitrate Lyase (1953)The glyoxylateThe glyoxylate bypass bypass (Revealed in 1957)(Revealed in 1957)lThe oxidation reactions of carbohydrate were not known despite of the success in understanding the chemical reactions of fermentation.
10、lCell free systems could NOT be successfully developed.lThe Wieland-Warburg controversy: Whether hydrogen or oxygen activation is the key to biological oxidation.lOxidizability of organic substances were systematically examined in isolated muscle tissues: rapid oxidation of salts of a number of acid
11、s (4-carbon dicarboxylic acid like succinate, malate, oxaloacetate, fumarate) was observed, but not linked to the oxidation of carbohydrates (Thunberg, 1910s-1920s).lSearched were carbon compounds that might serve as intermediates for pyruvate to be oxidized to CO2 and H2O.lSuspensions of minced pig
12、eon-breast muscle was found to be a good working system for studying respiration (Szent-Gyorgyi, 1934).FumaratelBoth fumaric acid and vitamin C were considered as hydrogen carriers between the foodstaff and O2.lNo six-carbon compounds yet revealed.lA linear, instead of cyclic, pathway was proposed.F
13、oodstaffFoodstafflCitrate was found to exhibit a similar catalytic effect as succinate on O2 consumption and pyruvate oxidation (1930s, Martius & Knoop).la-ketoglutarate were found to be a product of citrate oxidation (Martius and Knoop).lIsocitrate and cis-aconitate are oxidized as effectively as c
14、itrate by the suspension of minced pigeon muscle.lThe presence of malonate, a known inhibitor of the succinate dehydrogenase, caused the accumulation of citrate, a-ketoglurarate, in addition to succinate when fumarate was added.lAddition of pyruvate (derived from carbohydrate) and oxaloacetate led t
15、o accumulation of citrate in the medium.Krebs and Johnson (1937) “The role of citric acid in the intermediate metabolism in animal tissues. Enzymologica 4:148-156. Rejected by NatureUnknown substanceUnknown substanceKrebs and Johnson (1937) “The role of citric acid in the intermediate metabolism in
16、animal tissues. Enzymologica 4:148-156.Sir Hans Adolf Krebs(1990-1981) lPyruvate oxidation in bacteria depends on the presence of inorganic phosphate: proposed acetyl-phosphate as a key intermediate, serving as acetyl and phosphoryl donors.lActive acetylation of sulfonamide was detected in cell-free
17、 pigeon liver preparations, but not derived from acetyl-phosphate!lParticipation of a new coenzyme: disappeared on aging and dialysis; present in boiled extracts of all organs and microorganisms; could not be replaced by any known cofactor. lThe new coenzyme was purified and characterized (by Lipman
18、n and other labs): containing pantothenic acid (a known vitamin), adenine, phosphorus and sulfur (1940s-1950s).lStriking parallel was observed between CoA content and pyruvate utilization (mostly by others).lCoA-dependent citrate synthesis observed in pigeon liver faction and E. coli extract.Krebs d
19、iscovered the urea cycle in 1932before he elucidated the citric acid cycle! lAlso the fatty acid oxidation activities.lNo Glycolytic activitiesKennedy and Lehninger (1949) Oxidation of fatty acids and Tricarboxylic acid cycle intermediates by isolated rat liver mitochondria, JBC, 179: 957-972.O2-con
20、sumingbiological oxidationhas been associatedwith insoluble particulateportion of the cell.succinooxidase &cytochrome oxidasehave been found inmitochondria before. Apparent explanation: citrate is not an intermediate!lCitrate was synthesized from acetate, ATP, oxaloacetate in the presence of a solub
21、le enzyme preparation from animal tissues supplemented with E. coli extracts (providing the transacetylase activity), and CoA is required (1949-1951, Ochoa).Severo OchoaOrthophosphate is not needed; acetyl-phosphate is not an intermediate;Thiamine pyrophosphate (TPP) is required;With stoichiometric
22、production of SH groups;Acyl-mercaptide bond of acetyl-CoA is energy-rich.Severo Ochoala-lipoic acid was discovered as a “pyruvate oxidation factor” (Reed, Gunsalus, 1950s).lThe pyruvate and a-ketoglutarate oxidases were isolated from muscle and E. coli: MW being 2-5 million, each containing multipl
23、e molecules of lipoic acids and FAD (1951).lThe intermediates of the citric acid cycle are obligatory precursors of many biosynthesis, thus will be continuously removed.lCertain replenishment mechanism must exist.lIsocitrate lyase was first discovered in 1953 (Campbell et al, BBA 11:594): catalyzing
24、 the formation of glyoxylate and succinate when citrate or cis-aconitate was added into bacterial extracts.lMalate synthase was first revealed in 1956 (Wong and Ajl, JACS, 78:3230.): catalyzing the condensation of acetyl-CoA and glyoxylate to form malate.lKornberg, H. L.; Madsen, N. B. (1957) Synthe
25、sis of C4-dicarboxylic acids from acetate by a glyoxylate bypass of the tricarboxylic acid cycle, Biochimica et Biophysica Acta, 24, 651-3. lWhen cell-free extracts. of acetate-grown Pseudomonas KB 1 were incubated with C14H3CO2Na (I), ATP, coenzyme A, glutathione, and Na glyoxylate, malate was the
26、only labeled compound formed in the early stages of incubation. lWhen isocitrate replaced glyoxylate, malate was again the 1st labeled compound formed. lIn the absence of glyoxylate or isocitrate, no labeled compounds. were formed other than traces of acetyl CoA. lIt is concluded that Pseudomonas KB
27、 1, growing on acetate as sole source of C, possesses, in addition to the enzymic reactions of the tricarboxylic acid cycle, a mechanism offering an alternative route from isocitrate to malate, i.e., cleavage of isocitrate by isocitratase and the condensation of acetyl CoA and glyoxylate by malate s
28、ynthetase. lKornberg, H. L.; Krebs, H. A. j(1957) Synthesis of cell constituent from C2 units by a modified tricarboxylic acid cycle, Nature 179, 988-91. lOccurrence of a metabolic cycle in microorganisms which can derive all their C requirements from 2-C compds. lThe cycle is a variant of the trica
29、rboxylic acid cycle: acetate + oxaloacetate citrate cis-aconitate isocitrate succinate + glyoxylate; Glyoxylate + acetate malate, malate + 1/2O2 oxaloacetate. lAcetate reacts in the form of acetyl coenzyme A. The stages between citrate and malate in the tricarboxylic acid cycle are replaced by react
30、ion in which glyoxylate is a key metabolite, therefore, the cycle is referred to as the glyoxylate cycle. lMain discoveries leading to the elaboration of the cycle: (1) the finding that isocitrate, apart from undergoing dehydrogenation, is split enzymically to form succinate and glyoxylate; (2) the
31、recognition by Wong and Ajl of an enzyme system bringing about the synthesis of malate from glyoxylate and acetyl coenzyme A; (3) the demonstration of the ready occurrence of the combined action of the 2 enzyme systems in cell-free exts. The key reactions of the glyoxylate cycle have further been de
32、monstrated in Ricinus seedlings, accounting for the conversion of fat to carbohydrate. lPyruvate is converted to acetyl-CoAPyruvate is converted to acetyl-CoA by the action of by the action of pyruvate dehydrogenasepyruvate dehydrogenase complex complex, a huge enzyme , a huge enzyme plex.lAcetyl-Co
33、AAcetyl-CoA is converted to 2 CO is converted to 2 CO2 2 via the eight-step via the eight-step citric acid cyclecitric acid cycle, generating three NADH, one FADH, generating three NADH, one FADH2 2, , and one ATP (by substrate-level phophorylationand one ATP (by substrate-level phophorylation). ).l
34、Intermediates of citric acid cycle are also used as Intermediates of citric acid cycle are also used as biosynthetic precursorsbiosynthetic precursors for many other biomolecules, for many other biomolecules, including fatty acids, steroids, amino acids, heme, including fatty acids, steroids, amino
35、acids, heme, pyrimidinespyrimidines, and glucose., and glucose.lOxaloacetate can get replenished from pyruvate, via Oxaloacetate can get replenished from pyruvate, via a carboxylation reaction catalyzed by the biotin-a carboxylation reaction catalyzed by the biotin-containing pyruvate carboxylasecon
36、taining pyruvate carboxylase. .lThe activity of pyruvate dehydrogenase complex is The activity of pyruvate dehydrogenase complex is regulated by allosteric effectors and reversible regulated by allosteric effectors and reversible phosphorylationsphosphorylations. .lNet conversion of fatty acids to g
37、lucose can occur in Net conversion of fatty acids to glucose can occur in germinating seeds, some invertebrates and some germinating seeds, some invertebrates and some bacteria via the bacteria via the glycoxylateglycoxylate cycle cycle, which shares three , which shares three steps with the citric
38、acid cycle but bypasses the two steps with the citric acid cycle but bypasses the two decarboxylation steps, converting two molecules of decarboxylation steps, converting two molecules of acetyl-CoA to one succinateacetyl-CoA to one succinate. .lAcetyl-CoA (isocitrate) is partitioned into the glyoxylate Acetyl-CoA (isocitrate) is partitioned into the glyoxylate cycle and citric acid cycle via a coordinately regulation cycle and citric acid cycle via a coordinately regulation of the isocitrate dehydrogenase and isocitrate lyaseof the isocitrate dehydrogenase and isocitrate lyase. .
生物化學：Chapter 16 the Citric Acid Cycle (for Nov. 27, 2012)
