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29 Organic Chemistry William H. Brown & Christopher S. Foote 29-1 29 The Organic Chemistry of Metabolism Chapter 29 29-2 29 Introduction We have now studied the typical reactions of the major classes of organic compounds, and the structure and reactions of carbohydrates and lipids Now let us apply this background to the study of the organic chemistry of metabolism We study two key metabolic pathways • -oxidation of fatty acids • glycolysis 29-3 29 Five Key Participants Five of the compounds participating in these and a great many other metabolic pathways: • ATP, ADP, and AMP are universal carriers of phosphate groups • NAD+/NADH and FAD/FADH2 are coenzymes involved in the oxidation/reduction of metabolic intermediates Coenzyme: a low-molecular weight, nonprotein molecule or ion that binds reversibly to an enzyme, functions as a second substrate, and is regenerated by further reaction 29-4 29 Adenosine Triphosphate ATP is the most important compound involved in the transfer of phosphate groups phosphoric ester group O O N H2 N O - O-P- O-P- O-P- O-CH2 O O- O- OH H H phosphoric anhydride groups HO N N adenine N - N-glycosidic bond H OH -D-ribofuranose 29-5 29 Adenosine Triphosphate • hydrolysis of the terminal phosphate of ATP gives ADP and phosphate • in glycolysis, the phosphate acceptors are -OH groups of glucose and fructose O - O O O- P- O- P-O- A MP + H2 O O O- ATP Water (a phosphate acceptor) - O- P-O- A MP + H2 PO 4 O ADP 29-6 29 Nicotinamide adenine dinucleotide • a biological oxidizing agent O CNH2 + O - O-P-O-CH2 N O H H H H HO O OH a -N-glycosidic bond NH2 N O=P-O-CH2 - O N O H nicotinamide, N adenine N H H H HO OH 29-7 29 NAD+/NADH NAD+ is a two-electron oxidizing agent, and is reduced to NADH O CNH 2 H O CNH 2 H + H+ + 2 e : N+ Ad NAD+ (oxidized form) N Ad NADH (reduced form) 29-8 29 NAD+/NADH • NAD+ is involved in a variety of enzyme-catalyzed oxidation/reduction reactions; we deal with two of these in this chapter OH O C C H A secondary alcohol A ketone O C + 2 H+ + 2 e - O H + H2 O An aldehyde C OH + 2 H+ + 2 e - A carboxylic acid 29-9 29 NAD+/NADH -: H B e nzy m e B H O O C C O C-N H2 H + H H C-N H2 reduction oxidation O : N : H e nzy m e N Ad Ad NAD+ NADH 29-10 29 FAD/FADH2 O H3 C N H3 C N H H H H H N N H OH OH OH flavin O ribitol NH2 CH2 O N O O=P-O-P-O-CH2 O- O- N O H N adenine N H H H HO OH adenosine 29-11 29 FAD/FADH2 FAD participates in several types of enzymecatalyzed oxidation/reduction reactions • our concern is its participation in the oxidation of a CC bond in a fatty acid hydrocarbon chain to a C=C bond as shown in these balanced half-reactions Oxidation of the hydrocarbon chain: - CH 2 - CH2 - CH = CH - + 2 H+ + 2 e Reduction of FAD: FAD + 2 H+ + 2 e - FAD H2 29-12 29 FAD oxidation of C-C Enz Enz B: H H C R2 C R1 H H H3 C N H3 C N B H H R2 O C R1 C H H N N H O Ad H B Enz O H3 C N H3 C N N Ad H N H O -: B Enz 29-13 29 Fatty Acids and Energy Fatty acids in triglycerides are the principle storage form of energy for most organisms • carbon chains are in a highly reduced form • the energy yield per gram of fatty acid oxidized is greater than that per gram of carbohydrate Yield of Energy (kJ/mol) (kJ/g) C6 H1 2 O6 + 6 O 2 6 CO 2 + 6 H 2 O -2870 -15.9 Glucose CH3 ( CH2 ) 1 4 COOH + 2 3 O 2 1 6 CO 2 + 1 6 H 2 O -9791 -38.9 Palmitic acid 29-14 29 Oxidation of Fatty Acids There are two major stages in the oxidation of fatty acids • activation of the free fatty acid in the cytoplasm and its transport across the inner mitochondrial membrane • -oxidation -Oxidation: a series of four enzyme-catalyzed reactions that cleaves carbon atoms two at a time, from the carboxyl end of a fatty acids 29-15 29 Activation of Fatty Acids • activation begins in the cytoplasm with formation of a thioester between the carboxyl group of the fatty acid and the sulfhydryl group of coenzyme A • formation of the acyl-CoA derivative is coupled with the hydrolysis of ATP to AMP and pyrophosphate O R- C-O - A TP + HS-CoA Fatty acid Coenzyme A (as anion) A MP + P2 O 7 4 - O R- C-S- CoA + OH- An acyl-CoA derivative 29-16 29 -Oxidation • Reaction 1: stereospecific oxidation of a carboncarbon single bond a, to the carbonyl group O O a R- CH2 -CH2 - C-SCo A + FAD H C-SCo A C + FAD H2 C R H A trans-enoyl-CoA An acyl-CoA • Reaction 2: regiospecific and stereospecific hydration of the carbon-carbon double bond; only the Renantiomer is formed O H OH C-SCo A C C R H A trans-enoyl-CoA + H2 O C O H CH2 -C- SCoA R (R)--Hydroxyacyl-CoA 29-17 29 -Oxidation • Reaction 3: oxidation of the -hydroxyl group OH C H O CH2 -C- SCoA R (R)--Hydroxyacyl-CoA + N AD + O O R- C-CH2 - C- SCoA + N AD H -Ketoacyl-CoA • Reaction 4: cleavage of the carbon chain by a reverse Claisen condensation O O R- C-CH2 - C- SCoA -Ketoacyl-CoA O + CoA -SH Coenzyme A R- C-SCo A An acyl-CoA O + CH3 C-SCo A Acetyl-CoA 29-18 29 -Oxidation • mechanism of the reverse Claisen condensation O O R- C-CH2 - C- SCoA - S-Enz O- O R- C-CH2 - C- SCoA S- Enz Tetrahedral carbonyl addition intermediate O- O R- C-S- Enz An enzymethioester + CH2 = C-SCo A Enolate anion of acetyl-CoA 29-19 29 -Oxidation • this series of reactions is then repeated on the shortened fatty acyl chain and continues until the entire fatty acid chain is degraded to acetyl-CoA O CH3 ( CH2 ) 1 4 COH Hexadecanoic acid (Palmitic acid) 8 CoA -SH + A TP A MP + P2 O 7 4 - 7 N AD + 7 FAD O 8 CH3 CSCoA + Acetyl coenzyme A 7 N AD H 7 FAD H2 29-20 29 Glycolysis Glycolysis: from the Greek, glyko, sweet, and lysis, splitting • a series of 10 enzyme-catalyzed reactions by which glucose is oxidized to two molecules of pyruvate O C6 H1 2 O6 Glucose glycolysis 2 CH3 CCOO ten enzymecatalyzed steps Pyruvate + 6 H+ 29-21 29 Glycolysis - Rexn 1 • phosphorylation of a-D-glucose HO HO CH2 OH O O + OH a-D-Glucose - O hexokinase O- P-O- P-O- AMP Mg 2 + O- O- OH ATP CH2 OPO 3 2 O HO HO O + - O- P-O- AMP OH a-D-Glucose 6-phosphate OOH ADP 29-22 29 Glycolysis - Rexn 2 • isomerization of glucose 6-phosphate to fructose 6phosphate 6 HO HO CH2 OPO 3 2 O 2 OH phosphoglucoisomerase 1 OH a-D-Glucose 6-phosphate 6 CH2 OPO 3 2 1 CH2 OH O H HO 2 H OH ( a) H HO a-D-Fructose 6-phosphate 29-23 29 Glycolysis - Rexn 2 • this isomerization is most easily seen by considering the open-chain forms of each monosaccharide CHO H C OH CH2 OH C C HO H O H OH H OH H HO H OH H OH HO H OH H OH H OH H OH CH2 OPO 3 2 Glucose 6-phosphate (an aldohexose phosphate) CH2 OPO 3 2 An enediol CH2 OPO 3 2 Fructose 6-phosphate (a 2-ketohexose phosphate) 29-24 29 Glycolysis - Rexn 3 • phosphorylation of fructose 6-phosphate CH2 OPO 3 2 - CH2 OH C O HO H H OH H + A TP OH CH2 OPO 3 2 - Fructose 6-phosphate phosphofructokinase Mg 2+ C O HO H H OH H + A DP OH CH2 OPO 3 2 - Fructose 1,6-bisphosphate 29-25 29 Glycolysis - Rexn 4 • cleavage of fructose 6-phosphate to two triose phosphates CH2 OPO 3 2 C O HO H H OH H CH2 OPO 3 2 C= O aldolase OH CH2 OPO 3 2 - Fructose 1,6-bisphosphate CH2 OH Dihydroxyacetone phosphate + H C= O H C OH Glyceraldehyde 3-phosphate CH2 OPO 3 2 - 29-26 29 Glycolysis - Rexn 4 • reaction 4 is a reverse aldol reaction • a key intermediate is an imine formed by the C=O group of fructose 1,6-bisphosphate and an -NH2 group of the enzyme catalyzing this reaction CH2 OPO 3 2 C O HO H H OH H + + H 3 N Enz OH CH2 OPO 3 2 - Fructose 1,6-bisphosphate : B- ( - H2 O) CH2 OPO 3 2 + C= NH Enz HO H :BH O H H OH CH2 OPO 3 2 - Protonated imine 29-27 29 Glycolysis - Rexn 4 • reverse aldol reaction gives two three-carbon fragments, one as an imine 2- H OH CH2 OPO 3 2- Protonated imine : CH2 OPO 3 2 + C= NH Enz (enzyme-catalyzed reverse aldol HO H reaction) :BH O H CH2 OPO 3 C-N H Enz CHOH B + H H C= O H C OH CH2 OPO 3 2 - Glyceraldehyde 3-phosphate 29-28 29 Glycolysis - Rexn 4 • hydrolysis of the imine gives dihydroxyacetone phosphate and regenerates the -NH2 group of the enzyme CH2 OPO 3 2 - : C-N H Enz CHOH B H CH2 OPO 3 2 - + C= NH Enz CH2 OH :B- Protonated imine + H2 O CH2 OPO 3 2 C= O CH2 OH + + H 3 N Enz :B - Dihydroxyacetone phosphate 29-29 29 Glycolysis - Rexn 5 • isomerization of triose phosphates CH2 OH C=O CH2 OPO3 2 - CHOH C-OH CH2 OPO3 2 - Dihydroxyacetone phosphate An enediol intermediate CHO H C OH CH2 OPO3 2 Glyceraldehyde 3-phosphate 29-30 29 Glycolysis - Rexn 6 • oxidation of the -CHO group of glyceraldehyde 3phosphate • the -CHO group is oxidized to a carboxyl group • which is in turn converted to a carboxylic-phosphoric mixed anhydride • the oxidizing agent is NAD+, which is reduced to NADH A two-electron oxidation O G- C- H + H2 O A two-electron reduction N AD + + H+ + 2 e - O G- C- OH + 2 H+ + 2 e - N AD H 29-31 29 Glycolysis - Rexn 6 We divide this reaction into three stages • stage 1: formation of a thiohemiacetal O OH G- C- H + HS-Enz G- C- S- Enz H A thiohemiacetal • stage 2: oxidation of the thiohemiacetal by NAD+ H O O G- C- S- Enz G- C- S- Enz O H H H CNH 2 an enzyme-bound thioester O CNH 2 : N+ Ad N Ad 29-32 29 Glycolysis - Rexn 6 • stage 3: conversion of the thioester to a mixed anhydride G- C- S- Enz + -: O- P-OH O- : O O O- O G- C- O-P- OH Enz- S OO O G- C- O-P- OH + Enz -S: - O1,3-Bisphosphoglycerate (a mixed anhydride) 29-33 29 Glycolysis - Rexn 7 • transfer of a phosphate group from 1,3bisphosphoglycerate to ADP O 2C-OPO3 phosphoO glycerate kinase + O-P-O-AMP H C OH Mg2 + 2O CH2 OPO3 ADP 1,3-Bisphosphoglycerate COOO O + -O-P-O-P-O-AMP H C OH O- O2CH2 OPO3 ATP 3-Phosphoglycerate 29-34 29 Glycolysis - Rexn 8 • isomerization of 3-phosphoglycerate to 2phosphoglycerate COOH C OH CH2 OPO 3 2 3-Phosphoglycerate phosphoglycerate COOmutase H C OPO 3 2 CH2 OH 2-Phosphoglycerate 29-35 29 Glycolysis - Rexn 9 dehydration of 2-phosphoglycerate COOH C OPO 3 CH2 OH 2- enolase Mg2+ 2-Phosphoglycerate COOC OPO 3 2 - + H2 O CH2 Phosphoenolpyruvate 29-36 29 Glycolysis - Rexn 10 • phosphate transfer to ADP - COO pyruvate kinase Mg2+ C OPO 3 2 CH2 ADP Phosphoenolpyruvate ATP COO- COO- C-OH C= O CH2 CH3 Enol of pyruvate Pyruvate 29-37 29 Glycolysis • Summing these 10 reactions gives the net equation for glycolysis C6 H1 2 O6 + 2 NA D+ + 2 HPO 4 2 - + 2 ADP glycolysis Glucose O 2 CH3 CCOO - + 2 NADH + 2 ATP + 2 H 2 O Pyruvate 29-38 29 Fates of Pyruvate Pyruvate does not accumulate in cells, but rather undergoes one of three enzyme-catalyzed reactions, depending of the type of cell and its state of oxygenation • reduction to lactate • reduction to ethanol • oxidation and decarboxylation to acetyl-CoA A key to understanding the biochemical logic behind two of these fates is to recognize that glycolysis needs a continuing supply of NAD+ • if no oxygen is present to reoxidize NADH to NAD+, then another way must be found to do it 29-39 29 Lactate Fermentation • in vertebrates under anaerobic conditions, the most important pathway for the regeneration of NAD+ is reduction of pyruvate to lactate O CH3 CCOO- + NADH + H3 O+ lactate dehydrogenase Pyruvate OH CH3 CHCOO- + NAD+ + H2 O Lactate 29-40 29 Pyruvate to Lactate • while lactate fermentation does allow glycolysis to continue, it increases the concentration of lactate and also of H+ in muscle tissue, as seen in this balanced half-reaction C6 H1 2 O6 + 2 H2 O Glucose lactate fermentation OH 2 CH3 CHCOO- + 2 H3 O+ Lactate • when blood lactate reaches about 0.4 mg/100 mL, muscle tissue becomes almost completely exhausted 29-41 29 Pyruvate to Ethanol Yeasts and several other organisms regenerate NAD+ by this two step pathway • decarboxylation of pyruvate to acetaldehyde pyruvate O decarboxylase CH3 CCO2 - + H+ Pyruvate O CH3 CH + CO 2 Acetaldehyde • reduction of acetaldehyde to ethanol O CH3 CH + N AD H + H+ Acetaldehyde alcohol dehydrogenase CH3 CH2 OH + NA D + Ethanol 29-42 29 Pyruvate to Acetyl-CoA Under aerobic conditions pyruvate undergoes oxidative decarboxylation • the carboxylate group is converted to CO2 • the remaining two carbons are converted to the acetyl group of acetyl-CoA oxidative O decarboxylation CH3 CCOO- + NAD+ + CoASH Pyruvate O CH3 CSCoA + CO2 + NADH Acetyl-CoA 29-43 29 Pyruvate to Acetyl-CoA Oxidative decarboxylation of pyruvate to acetylCoA is considerably more complex than the previous equation suggests In addition to NAD+ (from the vitamin niacin) and coenzyme A (from the vitamin pantothenic acid), it also requires • FAD (from the vitamin riboflavin) • pyridoxal phosphate (from the vitamin pyridoxine) • lipoic acid 29-44 29 Pyruvate to Acetyl-CoA NH2 N N S O O O-P-O-P-OO- O- N Thiamine pyrophosphate H COO- S S Lipoic acid (as the carboxylate anion) 29-45 29 Prob 29.21 Describe the type of reaction involved in this biochemical synthesis of -hydroxybutyrate. O O 2 CH3 CSCoA Acetyl-CoA CoA-SH O CH3 CSCoA CoA-SH O SCoA Acetoacetyl-CoA 1 2 CO 2 O OH3 C OH O - SCoA (S)-3-Hydroxy-3methylglutaryl-CoA O CH3 CSCoA O O H+ 4 - 3 O Acetoacetate NADH 5 NAD+ O Acetone OH O O-Hydroxybutyrate 29-46 29 The Organic Chemistry of Metabolism End Chapter 29 29-47