Welcome to Class 9! Class 9: Outline and Objectives. Introductory Biochemistry!

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1 Welcome to Class 9 Introductory Biochemistry Class 9: Outline and Objectives l Pyruvate decarboxylase reaction l Thiamine pyrophosphate (TPP) l Pyruvate dehydrogenase reaction l Coenzymes l Reaction l Regulation l Citric acid cycle l Steps of the citric acid cycle l Prochirality l Regulation of the citric acid cycle l Role of the citric acid cycle in anabolism l Anaplerotic reactions l Pyruvate carboxylase and biotin l Glyoxylate cycle 1

2 The cofactor Thiamin PyroPhosphate is involved in reactions in which bonds to carbonyl carbons are synthesized or cleaved. Aerobic and anaerobic pathways: fates of pyruvate 2

3 Alcohol fermentation Pg 565 TPP: A coenzyme used in pyruvate decarboxylation Two of the three carbon atoms derived from pyruvate are carried transiently on TPP in the form of a hydroxyethyl group. Thiamine = Vitamin B 1 Pyruvate CO 2 Figure 14-15a, b 3

4 TPP reaction mechanism Figure 14-15c Three stages of cellular respiration Glycolysis splits sugars and partially oxidizes the products, generating substrates for complete oxidation to CO 2, and also generates some ATP and reduced cofactors. (In fermentations, the reduced cofactors are re-oxidized in non-energy-generating reactions.) The citric acid cycle completely oxidizes the products of glycolysis to CO 2 and generates reduced cofactors as well as some ATP. Respiratory re-oxidation of the reduced cofactors generated in the above stages is coupled to the synthesis of large amounts of ATP. Figure

5 Cell biology: The mitochondrion DNA Crista Matrix Glycolysis occurs in the cytoplasm. The citrate cycle takes place in the mitochondrial matrix. In non-photosynthetic eukaryotes, the mitochondrion is the site of most energy-yielding oxidative reactions and of the coupled synthesis of ATP. Pyruvate has to be transported from the cytosol into the mitochondrion (symport with H + ). Ribosomes Inner membrane Outer membrane Figures 19-2, 24-6 Pyruvate dehydrogenase reaction Very complex reaction: three different enzymes and five different cofactors participate. Irreversible, oxidative decarboxylation. Figure

6 Coenzyme A (Vitamin B 5 ) D-Ribose 3 -phosphate Acetyl-CoA: a thioester Figure 16-3 Pyridine Nucleotide Coenzymes Niacin (vitamin B 3 ) Nicotinamide ring Nicotinamide Adenine Dinucleotide (NAD) NAD + = oxidized form NADH = reduced form Figure 13-24a 6

7 Flavin nucleotide cofactors Riboflavin (vitamin B 2 ) Flavin Adenine Dinucleotide (FAD) FAD = oxidized form FADH 2 = reduced form Figure Similarities and differences Pyrophosphate Ribose ATP NADH Adenine FAD Coenzyme A 7

8 Lipoic acid A thioester (Not a vitamin for humans; we can synthesize it) Figure 16-4 The PDH complex The PDH complex from mammals is about 50nm in diameter - more than 5 times the size of a ribosome. E. coli PDH: (E 1 ) 24 (E 2 ) 24 (E 3 ) 12 plus regulatory proteins M r 5 x 10 6 Figure 16-5a,b 8

9 Pyruvate dehydrogenase reaction 1 Decarboxylation of pyruvate and transfer of active acetaldehyde to TPP 2 Oxidation of the hydroxyethyl group to a carboxyl group occurs at this step 3 Transacetylation 4 Re-oxidation of dihydrolipoate 5 Re-oxidation of FADH 2 Figure 16-6 Electron transfer in the pyruvate dehydrogenase reaction Dihydrolipoate + FAD Lipoate + FADH 2 FADH 2 + NAD + NADH + H + + FAD ΔE' º = ( 0.290) = V ΔG' º = 13.7 kj/mol ΔE' º = ( 0.219) = V ΔG' º = 19.5 kj/mol Dihydrolipoate + NAD + Lipoate + NADH + H + ΔE' º = ( 0.290) = V ΔG' º = 5.8 kj/mol (Even though FAD has a more positive standard reduction potential than NAD +, this applies only to free FAD; enzyme-bound FAD can have a more negative (or positive) reduction potential than free FAD.) 9

10 Pyruvate dehydrogenase Mechanistic advantages of multienzyme complexes: l Substrates have to diffuse only a short distance rate enhancement. l Substrate channeling increases specificity and minimizes side reactions. l Reactions catalyzed by a multienzyme complex may be coordinately controlled. E. coli PDH: (E 1 ) 24 (E 2 ) 24 (E 3 ) 12 plus regulatory proteins M r 5 x 10 6 Figure 16-5b Regulation of PDH Product inhibition by NADH and acetyl-coa (competitive inhibition), and inhibition by fatty acids. Stimulation by NAD + and free CoA. Reversible covalent modification by (de)phosphorylation of the E 1 subunit (occurs only in eukaryotic PDH). Phosphorylation inactivates the subunit. The PDH kinase is activated by ATP, NADH, and acetyl-coa. It is inhibited by pyruvate and ADP. Provides enzymatic leverage for the effects of these compounds. PDH kinase and PDH phosphatase are also part of the multienzyme complex. 10

11 Citric Acid Cycle Also called the tricarboxylic acid (TCA) cycle and the Krebs cycle Figure 16-7 The condensation reaction Figure

12 Formation of citrate 1 The concentration of oxaloacetate, which is regenerated in the cycle, is extremely low. Therefore, it is important that the citrate synthase reaction is very exergonic. This is achieved by the large negative G' o for acetyl-coa thioester hydrolysis. Acetyl-CoA has a high acyl-group transfer potential Accounts for virtually all of the driving force for citrate synthesis. Figures 13-16,

13 Isocitrate formation Figure 16-7 Isocitrate formation 2 The reaction is endergonic, but is pulled to the right because isocitrate is removed in step 3. Iron-sulfur cluster in the active site of aconitase Figure

14 Oxidative decarboxylation steps Figure 16-7 Oxidative decarboxylation steps 3 Step 3 is effectively irreversible at physiological CO 2 concentrations. 4 TPP, lipoate, FAD Step 4 is identical to the PDH reaction mechanism. The two enzyme complexes are likely to be related evolutionarily. Both decarboxylations are strongly exergonic. 14

15 Lipoic acid (Not a vitamin for humans; we can synthesize it) A thioester Arsenite (AsO2 ) can covalently bind to sulfhydryl compounds, such as lipoate. Inhibition of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase brings respiration to a halt. Figure 16-4 Substrate level phosphorylation Figure

16 Substrate-level phosphorylation 5 As for citrate synthase, the free energy of thioester hydrolysis drives the reaction. Many bacteria use ADP/ATP instead of GDP/GTP. Synthetases catalyze condensations that use nucleoside triphosphates (NTPs, e.g., ATP) as a source of energy for the synthetic reaction. Synthases (for example citrate synthase) do not require NTPs. Alkyl chain oxidation Figure

17 Three-step mechanism to oxidize an alkyl chain (A common theme in metabolic pathways) Dehydrogenation (oxidation) Step 6: Succinate dehydrogenase Hydration of the double bond Step 7: Fumarase Second oxidation Step 8: Malate dehydrogenase Succinate dehydrogenase (SDH) 6 SDH is a membrane-bound enzyme which has FAD bound covalently. Only the trans product fumarate is formed (the cis compound is named maleate). Malonate is a competitive inhibitor. It is unreactive as it cannot lose two hydrogens. 17

18 Hydration of fumarate to form malate 7 Fumarase is highly stereospecific: Only fumarate and L-malate are substrates. Formation of oxaloacetate 8 The reaction is endergonic, so the concentration of oxaloacetate would be much lower than that of L-malate at equilibrium. However, the product is effectively removed in step 1 of the cycle, which has a high affinity for oxaloacetate and is highly exergonic (ΔG = 32.2 kj/mol). 18

19 Where is the energy after one turn of the cycle? Where are the electrons after one turn of the cycle? Figure Overall energetics of pyruvate oxidation in the citric acid cycle First, calculate the energy released on complete oxidation of pyruvate (values from Lehninger): Glucose (C 6 H 12 O 6 ) + 6 O 2 6 CO H 2 O ΔG' 0 = 2840 kj/mol 2 Pyruvate (C 3 H 4 O 3 ) + 2 NADH + 2 H + Glucose + 2 NAD + ΔG' 0 = 146 kj/mol 2 NAD H e 2 NADH E' o = V ΔG' 0 = kj/mol 2 H 2 O O H e E' o = V ΔG' 0 = kj/mol 2 Pyruvate + 5 O 2 6 CO H 2 O ΔG' 0 = kj/mol Then use the value for the complete oxidation of one pyruvate to calculate the overall energetics of the citric acid cycle: Pyruvate O 2 3 CO H 2 O ΔG' 0 = kj/mol GDP + P i GTP + H 2 O ΔG' kj/mol 4 NAD H e 4 NADH E' o = V ΔG' 0 = kj/mol FAD + 2 H e FADH 2 E' o = V ΔG' 0 = 42.3 kj/mol 5 H 2 O 2.5 O H e E' o = V ΔG' 0 = kj/mol Pyruvate + 4 NAD + + FAD + 2 H 2 O + GDP + P i 3 CO NADH + FADH 2 + GTP ΔG' 0 = 20.6 kj/mol Of the energy released in the complete oxidation of one pyruvate, 30.5/ = 2.7% is captured as GTP (or ATP) and another ( )/ = 95% is potentially obtainable from re-oxidation of NADH and FADH 2. 19

20 Note that the two carbon atoms released as CO 2 are not the same two that enter as acetate. A Biochemical Puzzle But, how can that be? Citrate is a symmetrical molecule. Figure 16-7 The results of radiolabeling experiments like this one were initially very puzzling. The initial conclusion from this experiment was that citrate can not be an intermediate in the pathway Box 16-3, Figure 1 20

21 Citrate is a prochiral molecule A B A B The central C atom is called prochiral, because if one of its carboxymethyl substituents were converted into a different group, the molecule would become chiral. (Recall that this is also true for glycerol in the formation of L-glycerol 3-P from glycerol for glycerolipid biosynthesis or D-glyceraldehyde 3-P from dihydroxyacetone-p for glycolysis.) Citrate has the potential to react asymmetrically if the enzyme active site is asymmetric. Box 16-3, Figure 2 Isocitrate formation 2 A B A B A B A B Citrate is bound by aconitase with the two prochiral bonds pointing in different directions. Only one of the bound carbon atoms can react. Iron-sulfur cluster in the active site of aconitase Figure

22 Regulation of the citric acid cycle Substrate availability Product inhibition Allosteric feedback modulation Energy status (ATP/ADP/ AMP) Redox status (NAD+/NADH) Internal balance (acetylcoa, succinyl-coa, citrate Availability of alternative substrates Figure PFK-1 regulation Citrate inhibits PFK-1 allosterically. The rate of glycolysis is matched to the rate of the citric acid cycle by citrate. Figure 14-2a 22

23 Why are there so many steps to oxidize acetate? Oxidation to two CO 2 molecules occurs in two-electron steps The citrate cycle is the hub of intermediary metabolism: Intermediates and end products of catabolic pathways feed into the cycle. The citrate cycle is an amphibolic pathway; it also provides precursors for many biosynthetic pathways. It is both catabolic and anabolic. Note that the same number of carbon atoms are released as CO 2 as the number that enter as acetate. Every molecule that leaves the cycle as a precursor to another compound must be replaced. Figure

24 Why are there so many steps to oxidize acetate? Oxidation to two CO 2 molecules occurs in two-electron steps. The citrate cycle is the hub of intermediary metabolism: Intermediates and end products of catabolic pathways feed into the cycle. The citrate cycle is an amphibolic pathway; it also provides precursors for many biosynthetic pathways. Cycle intermediates that are removed for biosynthetic processes need to be replenished. This is done in anaplerotic reactions (anaplerotic = filling up). It is not necessary to replenish all intermediates, as they are part of a cycle. Replenishing even one of them will eventually lead to increased levels of the others, too. The citric acid cycle in anabolism Porphyrins, heme (plants, most bacteria) (animals, fungi) Figure

25 Anaplerotic reactions These anaplerotic reactions all convert a three-carbon molecule, which would otherwise be converted to acetyl-coa and then into CO 2, into a four-carbon molecule, which is added to the pool of citric acid cycle intermediates. The reactions are carboxylations. Biotin and the pyruvate carboxylase reaction Biotin (vitamin H or aka B 7 ) carries one-carbon groups in the form of activated CO 2. Figure

26 Biotin, lipoate and pantothenate are biological tethers Figure Class 9: Outline and Objectives l Pyruvate decarboxylase reaction l Thiamine pyrophosphate (TPP) l Pyruvate dehydrogenase reaction l Coenzymes l Reaction l Regulation l Citric acid cycle l Steps of the citric acid cycle l Prochirality l Regulation of the citric acid cycle l Role of the citric acid cycle in anabolism l Anaplerotic reactions l Pyruvate carboxylase and biotin l Glyoxylate cycle 26

27 Living on Fat Vertebrates can t survive on a primarily lipid diet because they cannot convert acetate (as acetyl-coa), the major product of fatty acid catabolism, into three- or four-carbon molecules. This means that they cannot use acetate to replenish the citric acid cycle pool or to form carbohydrates or molecules derived from these. The glyoxylate cycle Plants and many invertebrates and microorganisms can use fatty acids as their sole carbon source. They have the ability to convert the two-carbon compound acetate (as acetyl-coa) into the four-carbon citric acid cycle intermediate succinate to replenish the pool. The citric acid cycle intermediates can also be converted subsequently into three-carbon carbohydrate precursors. Net reaction: 2 Acetyl-CoA + NAD H 2 O Succinate + 2 CoA + NADH + H + 27

28 The glyoxylate cycle Occurs in plants and some invertebrates and microorganisms. Requires two unique enzymes, isocitrate lyase and malate synthase. The two-carbon compound glyoxylate is produced by the first enzyme and combined with acetate by the second enzyme. Net reaction: 2 Acetyl-CoA + NAD H 2 O Succinate + 2 CoA + NADH + H + OAA PEP Carbohydrates Figure Relationship between glyoxylate and citrate cycles Figure 16-23,24 28

29 The citric acid cycle occupies a central position in cellular metabolism. The citric acid cycle occupies a central position in cellular metabolism. 29

30 Where are the electrons after one turn of the cycle? Figure Three stages of cellular respiration Glycolysis splits sugars and partially oxidizes the products, generating substrates for complete oxidation to CO 2, and also generates some ATP and reduced cofactors. (In fermentations, the reduced cofactors are re-oxidized in non-energy-generating reactions.) The citric acid cycle completely oxidizes the products of glycolysis to CO 2 and generates reduced cofactors as well as some ATP. Respiratory re-oxidation of the reduced cofactors generated in the above stages is coupled to the synthesis of large amounts of ATP. Figure