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1、Chapter 16 The Citric Acid Cycle,The common pathway leading to the complete oxidation of carbohydrates, fatty acids, and amino acids to CO2 A pathway providing many precursors for biosynthetic reactions,Fates of pyruvate,Citric Acid Cycle (三羧酸循环),Also called the tricarboxylic acid cycle (TCA) or the
2、 Krebs cycle Pyruvate completely oxidized to CO2 and H2O in the presence of O2-cellular respiration Occurs in eight steps in mitochondria Energy efficiently conserved A hub in metabolism, serving in both catabolic and anabolic processes,1. The cellular respiration (complete oxidation of fuels) can b
3、e divided into three stages,Stage I All the fuel molecules are oxidized to generate a common two-carbon unit, acetyl-CoA. Stage II Acetyl-CoA is completely oxidized to CO2, with electrons collected by NAD and FAD via the citric acid cycle. Stage III Electrons of NADH and FADH2 are transferred to O2
4、via a series of carriers, producing H2O and a H+ gradient, which will promote ATP formation.,2. Pyruvate is oxidized to acetyl-CoA by the pyruvate dehydrogenase complex,Pyruvate is first transported into mitochondria via a specific transporter on the inner membrane. Pyruvate is converted to acetyl-C
5、oA and CO2 by oxidative decarboxylation. The pyruvate dehydrogenase (PDH) complex is a huge multimeric assembly of three kinds of enzymes, having 60 subunits in bacteria and more in mammals.,Production of acetyl-CoA,Oxidative decarboxylation,Critical to its role as an acyl carrier in a number of met
6、abolic reactions,(thioester),Lipoate can act as a carrier of both hydrogen and an acetyl group,Production of acetyl-CoA,Oxidative decarboxylation,Thiamine pyrophosphate (TPP, 硫胺焦磷酸, derived from vitamin B1) acts as the coenzyme of the decarboxylase.,Pyruvate Dehydrogenase Complex (PDH),Three-enzyme
7、complex E1: pyruvate dehydrogenase (TPP) E2: dihydrolipoyl transacetylase (lipoate)-core E3: dihydrolipoyl dehydrogenase (FAD) Plus 2 regulatory proteins (a protein kinase and a phosphoprotein phosphatase),Cryoelectron micrograph of PDH complexes isolated from bovine kidney,Three-dimensional image o
8、f the PDH complex,E2 consists of three types of domains linked by short polypeptide linkers,Oxidative decarboxylation of pyruvate to acetyl-CoA by the PDH complex,Substrates of the five reactions catalyzed by the pyruvate dehydrogenase complex are efficiently channeled-substrate channeling The long
9、lipoyllysine arm of E2 swings from the active site of E1 to E2 to E3, tethering the intermediates to the enzyme complex to allow substrate channeling. The multienzyme complexes catalyzing the oxidative decarboxylation of a few different kinds of a-keto acids, including pyruvate dehydrogenase complex
10、, a-ketoglutarate dehydrogenase complex and branched-chain a-keto acid dehydrogenase complex, show remarkable structure and function similarities (all have identical E3, similar E1 and E2).,3. The complete oxidation of pyruvate in animal tissues was proposed to undergo via a cyclic pathway,O2 consum
11、ption and pyruvate oxidation in minced muscle tissues were found to be stimulated by some four-carbon dicarboxylic acids (fumarate, succinate, malate and oxaloacetate), five-carbon dicarboxylic acid (a-ketoglutarate ), or six-carbon tricarboxylic acids (citrate, isocitrate, cis-aconitate). A small a
12、mount of any of these organic acids stimulates many folds of pyruvate oxidation!,Malonate inhibits pyruvate oxidation regardless of which active organic acid is added! Hans Krebs proposed the “citric acid cycle” for the complete oxidation of pyruvate in animal tissues in 1937. The citric acid cycle
13、was confirmed to be universal in cells by in vitro studies with purified enzymes and in vivo studies with radio isotopes (“radio isotope tracer experiments”). Krebs was awarded the Nobel Prize in medicine in 1953 for revealing the citric acid cycle (thus also called the Krebs cycle).,4. The acetyl g
14、roup (carried by CoA) is completely oxidized to CO2 via the citric acid cycle,The 4-carbon oxaloacetate (草酰乙酸) acts as the “carrier” for the oxidation. The two carbons released as 2 CO2 in the first cycle of oxidation are not from the acetyl-CoA just joined. The 8 electrons released are collected by
15、 three NAD+ and one FAD. One molecule of ATP (or GTP) is produced per cycle by substrate-level phosphorylation.,Reactions of the citric acid cycle,The citric acid cycle,Acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2H2O 2CO2 + 3NADH + FADH2 + GTP + 2H+ + CoA,Step 1: condensation,Structure of citrate synthas
16、e,Citrate synthase -induced fit,Step 2: dehydration & hydration,Step 3 oxidative decarboxylation,Two isozymes,Similar to pyruvate dehydrogenase complex,Step 4 oxidative decarboxylation,Step 5 substrate-level phosphorylation,(Two isozymes, one for GDP, the other for ADP),(Membrane-bound),Step 6 dehyd
17、rogenation,Competitive Inhibitor,Stereospecific,Step 7 hydration,(trans),(cis),Step 8 dehydrogenation,Products of one turn of the citric acid cycle,Products of one turn of the citric acid cycle,Reactions of the citric acid cycle,Irreversible,5. The complete oxidation of one glucose may yield as many
18、 as 32 ATP,All the NADH and FADH2 will eventually pass their electrons to O2 after being transferred through a series of electron carriers. The complete oxidation of each NADH molecule leads to the generation of about 2.5 ATP, and FADH2 of about 1.5 ATP. The overall efficiency of energy conservation
19、 is about 34% using the free energy changes under standard conditions and about 65% using actual free energy changes in cells.,6. The intermediates of the citric acid cycle are important sources for biosynthetic precursors,The citric acid cycle is the hub of intermediary metabolism serving both the
20、catabolic and anabolic processes (thus an amphibolic pathway). Any compounds that give rise to four- or five- carbon intermediates of the cycle can be oxidized by the cycle. It provides precursors for the biosynthesis of glucose, amino acids, nucleotides, fatty acids, heme groups, etc.,The carbon sk
21、eletons of amino acids are oxidized via the citric acid cycle (Fig. 18-15),Citric acid cycle provides precursors for many biosynthetic pathways,Intermediates of the citric acid cycle get replenished by anaplerotic reactions when consumed by biosynthesis. The most common anaplerotic reactions covert
22、either pyruvate or phosphoenolpyruvate to oxaloacetate or malate.,Pyruvate carboxylase catalyzes the carboxylation of pyruvate using covalently bound biotin (a vitamin) as the coenzyme. The long flexible arm of biotin switches between two active site of the pyruvate carboxylase (one for attaching a
23、HCO3- to biotin and the other for transferring the carboxyl group to pyruvate). Acetyl-CoA is a positive allosteric modulator for pyruvate carboxylase.,Biotin (a vitamin) plays a key role in the carboxylation reaction- specialized carrier of one-carbon groups,Some anaerobic bacteria, lacking a-ketog
24、lutarate dehydrogenase, make biosynthetic precursors via the incomplete citric acid cycle, which could be an early evolution stage of the citric acid cycle.,Regulation of the citric acid cycle,Regulatory enzymes: Pyruvate dehydrogenase complex Citrate synthase Isocitrate dehydrogenase a-ketoglutarat
25、e dehydrogenase,7. The pyruvate dehydrogenase complex in vertebrates is regulated allosterically and covalently,The formation of acetyl-CoA from pyruvate is a key irreversible step in animals because they are unable to convert acetyl-CoA to glucose. The complex (in all organisms) is allosterically i
26、nhibited by signaling molecules indicating a rich source of energy, e.g., ATP, acetyl-CoA, NADH, long chain fatty acids; activated by molecules indicating a lack (or demand) of energy, e.g., AMP, CoA, NAD+, Ca2+.,The activity of the complex (in vertebrates, probably also in plants, but not in E. col
27、i) is also regulated by reversible phosphorylation of one of the enzymes, E1, in the complex: phosphorylation of a specific Ser residue inhibits and dephosphorylation activates the complex. The kinase and phosphatase is also part of the enzyme complex. E1 (unphosphorylated)-active E1 (phosphorylated
28、)-inactive Kinase involved is activated by ATP, but inhibited by a drug called dichloroacetate.,8. The rate of the citric acid cycle is controlled at three exergonic irreversible steps,Regulatory enzymes: citrate synthase, isocitrate dehydrogenase and a-ketoglutarate dehydrogenase Inhibited by produ
29、ct feedback (citrate, succinyl-CoA) and high energy charge (ATP, NADH) Activated by a low energy charge (ADP) or a signal for energy requirement (Ca2+).,Regulation of the citric acid cycle,*,9. Net conversion of acetate to carbohydrates is achieved via the glyoxylate cycle,In vertebrates there is no
30、 net conversion of acetate (also from fatty acids and amino acids) to any of the citric acid cycle intermediate, thus neither to carbohydrates. Net conversion of acetate to four-carbon citric acid cycle intermediates occurs via the glyoxylate cycle, found in plants, certain invertebrates, and some m
31、icroorganisms (including E. coli and yeast).,The glyoxylate cycle,The glyoxylate cycle shares three steps and bypasses the rest, including the two decarboxylation steps, of the citric acid cycle. Two acetyl-CoA molecules enter each glyoxylate cycle with a net production of one succinate. Isocitrate
32、lyase and malate synthase are the two bypassing enzymes, converting isocitrate to malate via the glyoxylate intermediate, releasing a succinate on the way.,The glyoxylate cycle,Convert acetate to carbohydrate in plants, certain invertebrates and some microorganisms 2 Acetyl-CoA + NAD+ + 2H2O Succina
33、te + 2CoA + NADH + H+,Electron micrograph of a germinating cucumber seed,10. Conversion of fatty acids to glucose (in germinating seeds) occurs in three intracellular compartments,Fatty acids are first converted to acetyl-CoA, which is in turn converted to succinate via the glyoxylate cycle in glyox
34、ysomes. Succinate is transported to mitochondria and converted to malate via the citric acid cycle. Malate is then transported out of mitochondria and is converted to glucose in the cytosol.,Relationship between the citric acid cycle and the glyoxylate cycle,11. The partitioning of isocitrate, betwe
35、en the citric acid and glyoxylate cycles is coordinately regulated,The activity of the E. coli isocitrate dehydrogenase is inhibited when phosphorylated by a specific kinase and activated when dephosphorylated by a specific phosphatase.,The citric acid and glyoxylate cycles are coordinately regulate
36、d,The kinase and phosphatase activities are located in two domains of the same polypeptide and are reciprocally regulated: the kinase is allosterically inhibited (while the phosphatase activated) by molecules indicating an energy depletion, e.g., accumulation of intermediates of glycolysis and the c
37、itric acid cycle. The allosteric inhibitors of the kinase also act as inhibitors for the isocitrate lyase: i.e., they activate the dehydrogenase while simultaneously inhibit the lyase.,Key words,Pyruvate dehydrogenase complex-reaction, regulation The citric acid cycle-reaction, regulation The glyoxy
38、late cycle-products,Summary,Pyruvate, the product of glycolysis, is converted to acetyl-CoA, the starting material of the citric acid cycle, by the pyruvate dehydrogenase (PDH) complex. The PDH complex is composed of multiple copies of three enzymes and five different coenzymes. The citric acid cycl
39、e is a nearly universal central catabolic pathway in which compounds derived from the breakdown of carbohydrates, fats and proteins are oxidized to CO2, with most of the energy of oxidation temporarily held in the electron carriers NADH and FADH2.,For each acetyl-CoA oxidized by the citric acid cycl
40、e, the energy gain consists of three molecules of NADH, one FADH2, and one ATP or GTP. The overall rate of the citric acid cycle is controlled by the rate of conversion of pyruvate to acetyl-CoA and by the flux through citrate synthase, isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase. Th
41、e PDH complex is inhibited allosterically by metabolites that signal a sufficiency of metabolic energy (ATP, acetyl-CoA, NADH and fatty acids) and stimulated by metabolites that indicate a reduced energy supply (AMP, NAD+ and CoA).,The glyoxylate cycle is active in the germinating seeds of some plan
42、ts and in certain microorganisms that can live on acetate as the sole carbon source. In the glyoxylate cycle, the bypassing of the two decarboxylation steps of the citric acid cycle makes possible of the net formation of succinate. Vertebrates lack the glyoxylate cycle and cannot synthesize glucose
43、from acetate or the fatty acids that give rise to acetyl-CoA. The partitioning of isocitrate between the citrate acid cycle and the glyoxylate cycle is controlled at the level of isocitrate dehydrogenase, which is regulated by reversible phosphorylation.,References,Kay, J & Weitzman, P. D. (eds) (19
44、87) Krebs Citric Acid Cycle: Half a Century and Still Turning. Biochemical Society Symposium 54, The Biochemical Society, London. Harris, R. A., Bowker-Kinley, M. M., Huang, B., &Wu, P. (2002) Regulation of the activity of the pyruvate dehydrogenase complex. Adv. Enzyme Regul. 42, 249-259. Knowles,
45、J. (1989) The mechanism of biotin-dependent enzymes. Annu. Rev. Biochem. 272, 8105-8108.,Ovadi, J. & Srere, P. (2000) Macromolecular compartmentation and channeling. Int. Rev. Cytol. 192, 255-280. Hansford, R. G. (1980) Control of mitochondrial substrate oxidation. Curr. Top. Bioenerget. 10, 217-278. Eastmond, P. J. & Graham, I. A. (2001) Re-examining the role of the glyoxylate cycle in oilseeds. Trends Plant Sci. 6, 72-77.,
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