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Horton • Moran • Scrimgeour • Perry • Rawn Principles of Biochemistry Fourth Edition Chapter 7 Coenzymes and Vitamins Prentice Hall c2002 Chapter 7 Copyright © 2006 Pearson Prentice 1 Hall, Inc. Chapter 7 - Coenzymes and Vitamins • Some enzymes require cofactors for activity (1) Essential ions (mostly metal ions) (2) Coenzymes (organic compounds) Apoenzyme + Cofactor (protein only) Holoenzyme (active) (inactive) Prentice Hall c2002 Chapter 7 2 Coenzymes • Coenzymes act as group-transfer reagents • Hydrogen, electrons, or other groups can be transferred • Larger mobile metabolic groups can be attached at the reactive center of the coenzyme • Coenzyme reactions can be organized by their types of substrates and mechanisms Prentice Hall c2002 Chapter 7 3 Fig 7.1 Types of cofactors Prentice Hall c2002 Chapter 7 4 7.1 Many Enzymes Require Inorganic Cations • Enzymes requiring metal ions for full activity: (1) Metal-activated enzymes have an absolute requirement or are stimulated by metal ions (examples: K+, Ca2+, Mg2+) (2) Metalloenzymes contain firmly bound metal ions at the enzyme active sites (examples: iron, zinc, copper, cobalt ) Prentice Hall c2002 Chapter 7 5 Fig 7.2 Mechanism of carbonic anhydrase • Action of carbonic anhydrase, a metalloenzyme • Zinc ion promotes the ionization of bound H2O. Resulting nucleophilic OHattacks carbon of CO2 (continued next slide) Prentice Hall c2002 Chapter 7 6 Fig. 7.2 (continued) Prentice Hall c2002 Chapter 7 7 Iron in metalloenzymes • Iron undergoes reversible oxidation and reduction: Fe3+ + e- (reduced substrate) Fe2+ + (oxidized substrate) • Enzyme heme groups and cytochromes contain iron • Nonheme iron exists in iron-sulfur clusters (iron is bound by sulfide ions and S- groups from cysteines) • Iron-sulfur clusters can accept only one e- in a reaction Prentice Hall c2002 Chapter 7 8 Fig 7.3 Iron-sulfur clusters • Iron atoms are complexed with an equal number of sulfide ions (S2-) and with thiolate groups of Cys side chains Prentice Hall c2002 Chapter 7 9 7.2 Coenzyme Classification • There are two classes of coenzymes (1) Cosubstrates are altered during the reaction and regenerated by another enzyme (2) Prosthetic groups remain bound to the enzyme during the reaction, and may be covalently or tightly bound to enzyme Prentice Hall c2002 Chapter 7 10 Classification of coenzymes in mammals (1) Metabolite coenzymes - synthesized from common metabolites (2) Vitamin-derived coenzymes - derivatives of vitamins (vitamins cannot be synthesized by mammals, but must be obtained as nutrients) Prentice Hall c2002 Chapter 7 11 Prentice Hall c2002 Chapter 7 12 7.3 ATP and Other Nucleotide Cosubstrates • Nucleoside triphosphates are examples Fig 7.4 ATP Prentice Hall c2002 Chapter 7 13 Reactions of ATP • ATP is a versatile reactant that can donate its: (1) Phosphoryl group (g-phosphate) (2) Pyrophosphoryl group (g,b phosphates) (3) Adenylyl group (AMP) (4) Adenosyl group Prentice Hall c2002 Chapter 7 14 SAM synthesis • ATP is also a source of other metabolite coenzymes such as S-adenosylmethionine (SAM) • SAM donates methyl groups in many biosynthesis reactions Methionine + ATP Prentice Hall c2002 S-Adenosylmethionine + Pi + PPi Chapter 7 15 Fig 7.5 S-Adenosylmethionine • Activated methyl group in red Prentice Hall c2002 Chapter 7 16 S-Adenosylmethionine (SAM) is a methyl donor in many biosynthetic reactions • SAM donates the methyl group for the synthesis of the hormone epinephrine from norepinephrine Prentice Hall c2002 Chapter 7 17 Fig 7.6 • Nucleotide-sugar coenzymes are involved in carbohydrate metabolism • UDP-Glucose is a sugar coenzyme. It is formed from UTP and glucose 1-phosphate (UDP-glucose product next slide) Prentice Hall c2002 Chapter 7 18 Fig 7.6 (continued) Prentice Hall c2002 Chapter 7 19 Vitamin-Derived Coenzymes and Nutrition • Vitamins are required for coenzyme synthesis and must be obtained from nutrients • Animals rely on plants and microorganisms for vitamin sources (meat supplies vitamins also) • Most vitamins must be enzymatically transformed to the coenzyme Prentice Hall c2002 Chapter 7 20 Table 7.1 Vitamins, nutritional deficiency diseases Vitamin Disease Ascorbate (C) Nicotinic acid Riboflavin (B2) Pantothenate (B3) Thiamine (B1) Pyridoxal (B6) Biotin Folate Cobalamin (B12) Scurvy Pellagra Growth retardation Dermatitis in chickens Beriberi Dermatitis in rats Dermatitis in humans Anemia Pernicious anemia Prentice Hall c2002 Chapter 7 21 Box 7.1 Vitamin C: a vitamin but not a coenzyme • A reducing reagent for hydroxylation of collagen • Deficiency leads to the disease scurvy • Most animals (not primates) can synthesize Vit C Prentice Hall c2002 Chapter 7 22 7.4 NAD+ and NADP+ • Nicotinic acid (niacin) is precursor of NAD and NADP • Lack of niacin causes the disease pellagra • Humans obtain niacin from cereals, meat, legumes Prentice Hall c2002 Chapter 7 23 Fig 7.8 Oxidized, reduced forms of NAD (NADP) Prentice Hall c2002 Chapter 7 24 Box 7.2 NAD Binding to Dehydrogenases Prentice Hall c2002 Chapter 7 25 NAD and NADP are cosubstrates for dehydrogenases • Oxidation by pyridine nucleotides always occurs two electrons at a time • Dehydrogenases transfer a hydride ion (H:-) from a substrate to pyridine ring C-4 of NAD+ or NADP+ • The net reaction is: NAD(P)+ + 2e- + 2H+ Prentice Hall c2002 NAD(P)H + H+ Chapter 7 26 Ordered mechanism for lactate dehydrogenase • Reaction of lactate dehydrogenase • NAD+ is bound first and NADH released last Prentice Hall c2002 Chapter 7 27 Fig 7.9 Mechanism of lactate dehydrogenase • Hydride ion (H:-) is transferred from C-2 of L-lactate to the C-4 of NAD+ Prentice Hall c2002 Chapter 7 28 7.5 FAD and FMN • Flavin adenine dinucleotide (FAD) and Flavin mono-nucleotide (FMN) are derived from riboflavin (Vit B2) • Flavin coenzymes are involved in oxidationreduction reactions for many enzymes (flavoenzymes or flavoproteins) • FAD and FMN catalyze one or two electron transfers Prentice Hall c2002 Chapter 7 29 Fig 7.10 Riboflavin and its coenzymes (a) Riboflavin, (b) FMN (black), FAD (black/blue) Prentice Hall c2002 Chapter 7 30 Fig 7.11 Reduction, reoxidation of FMN or FAD Prentice Hall c2002 Chapter 7 31 7.6 Coenzyme A (CoA or HS-CoA) • Derived from the vitamin pantothenate (Vit B3) • Participates in acyl-group transfer reactions with carboxylic acids and fatty acids • CoA-dependent reactions include oxidation of fuel molecules and biosynthesis of carboxylic acids and fatty acids • Acyl groups are covalently attached to the -SH of CoA to form thioesters Prentice Hall c2002 Chapter 7 32 Fig 7.12 Coenzyme A Prentice Hall c2002 Chapter 7 33 Fig. 7.12 Acyl carrier protein Prentice Hall c2002 Chapter 7 34 Fig. 7.13 Acetyl CoA Prentice Hall c2002 Chapter 7 35 7.7 Thiamine Pyrophosphate (TPP) • TPP is a derivative of thiamine (Vit B1) • Reactive center is the thiazolium ring (with a very acidic hydrogen atom at C-2 position) • TPP participates in reactions of: (1) Decarboxylation (2) Oxidative decarboxylation (3) Transketolase enzyme reactions Prentice Hall c2002 Chapter 7 36 Fig 7.14 Thiamine (Vitamin B1) and TPP Prentice Hall c2002 Chapter 7 37 Fig 7.15 Mechanism of pyruvate dehydrogenase (3 slides) Prentice Hall c2002 Chapter 7 38 Fig 7.15 (continued) From previous slide Prentice Hall c2002 Chapter 7 39 Fig 7.15 (continued) From previous slide Prentice Hall c2002 Chapter 7 40 7.8 Pyridoxal Phosphate (PLP) • PLP is derived from Vit B6 family of vitamins (deficiencies lead to dermatitis and disorders of protein metabolism) • Vitamin B6 is phosphorylated to form PLP • PLP is a prosthetic group for enzymes catalyzing reactions involving amino acid metabolism (isomerizations, decarboxylations, side chain eliminations or replacements) Prentice Hall c2002 Chapter 7 41 Fig 7.16 B6 Vitamins and pyridoxal phosphate (PLP) Prentice Hall c2002 Chapter 7 42 Fig 7.17 Binding of substrate to a PLPdependent enzyme Prentice Hall c2002 Chapter 7 43 Fig. 7.17 (continued) From previous slide Prentice Hall c2002 Chapter 7 44 Fig 7.18 Mechanism of transaminases (5 slides) Prentice Hall c2002 Chapter 7 45 Fig 7.18 (continued) Prentice Hall c2002 Chapter 7 46 Fig 7.18 (continued) Prentice Hall c2002 Chapter 7 47 Fig 7.18 (continued) Prentice Hall c2002 Chapter 7 48 Fig 7.18 (continued) Prentice Hall c2002 Chapter 7 49 7.9 Biotin • Biotin is required in very small amounts because it is available from intestinal bacteria • Avidin (raw egg protein) binds biotin very tightly and may lead to a biotin deficiency (cooking eggs denatures avidin so it does not bind biotin) • Biotin (a prosthetic group) enzymes catalyze: (1) Carboxyl-group transfer reactions (2) ATP-dependent carboxylation reactions Prentice Hall c2002 Chapter 7 50 Fig 7.19 Enzyme-bound biotin • Biotin is linked by an amide bond to the e-amino group of a lysine residue of the enzyme • The reactive center of biotin is the N-1 (red) Prentice Hall c2002 Chapter 7 51 Fig 7.20 Reaction catalyzed by pyruvate carboxylase Two step mechanism (next slide) Step 1: Formation of carboxybiotin-enzyme complex (requires ATP) Step 2: Enolate form of pyruvate attacks the carboxyl group of carboxybiotin forming oxaloacetate and regenerating biotin Prentice Hall c2002 Chapter 7 52 Prentice Hall c2002 Chapter 7 53 7.10 Tetrahydrofolate (THF) • Vitamin folate is found in green leaves, liver, yeast • The coenzyme THF is a folate derivative where positions 5,6,7,8 of the pterin ring are reduced • THF contains 5-6 glutamate residues which facilitate binding of the coenzyme to enzymes • THF participates in transfers of one carbon units at the oxidation levels of methanol (CH3OH), formaldehyde (HCHO), formic acid (HCOOH) Prentice Hall c2002 Chapter 7 54 Fig 7.21 Pterin, folate and tetrahydrofolate (THF) Prentice Hall c2002 Chapter 7 55 Formation of tetrahydrofolate (THF) from folate Prentice Hall c2002 Chapter 7 56 Fig 7.22 • One-carbon derivatives of THF Continued next slide Prentice Hall c2002 Chapter 7 57 Fig 7.22 (continued) Prentice Hall c2002 Chapter 7 58 Fig. 7.23 5,6,7,8, Tetrahydrobiopterin, a pterin coenzyme • Coenzyme has a 3-carbon side chain at C-6 • Not vitamin-derived, but synthesized by some organisms Prentice Hall c2002 Chapter 7 59 7.11 Cobalamin (Vitamin B12) • Coenzymes: methylcobalamin, adenosylcobalamin • Cobalamin contains a corrin ring system and a cobalt (it is synthesized by only a few microorganisms) • Humans obtain cobalamin from foods of animal origin (deficiency leads to pernicious anemia) • Coenzymes participate in enzyme-catalyzed molecular rearrangements in which an H atom and a second group on the substrate exchange places Prentice Hall c2002 Chapter 7 60 Fig 7.24 Cobalamin (Vit B12) and its coenzymes (a) Cobalamin. Corrin ring (black) Prentice Hall c2002 Chapter 7 61 Fig 7.24 (continued) (b) Abbreviated structure of cobalamin coenzymes Prentice Hall c2002 Chapter 7 62 Fig 7.25 Intramolecular rearrangements catalyzed by adenosylcobalamin enzymes (a) Rearrangement of an H and substituent X on an adjacent carbon Prentice Hall c2002 Chapter 7 63 Fig. 7.25 (continued) (b) Rearrangement of methylmalonyl CoA Prentice Hall c2002 Chapter 7 64 Methylcobalamin participates in the transfer of methyl groups Prentice Hall c2002 Chapter 7 65 7.12 Lipoamide • Coenzyme lipoamide is the protein-bound form of lipoic acid • Animals can synthesize lipoic acid, it is not a vitamin • Lipoic acid is an 8-carbon carboxylic acid with sulfhydryl groups on C-6 and C-8 • Lipoamide functions as a “swinging arm” that carries acyl groups between active sites in multienzyme complexes Prentice Hall c2002 Chapter 7 66 Fig 7.26 Lipoamide • Lipoic acid is bound via an amide linkage to the e-amino group of an enzyme lysine • Reactive center of the coenzyme shown in red Prentice Hall c2002 Chapter 7 67 Transfer of an acyl group between active sites • Acetyl groups attached to the C-8 of lipoamide can be transferred to acceptor molecules • In the pyruvate dehydrogenase reaction the acetyl group is transferred to coenzyme A to form acetylSCoA Prentice Hall c2002 Chapter 7 68 7.13 Lipid Vitamins • Four lipid vitamins: A, D, E, K • All contain rings and long, aliphatic side chains • All are highly hydrophobic • The lipid vitamins differ widely in their functions Prentice Hall c2002 Chapter 7 69 A. Vitamin A (Retinol) • Vit A is obtained from liver, egg yolks, milk products or b-carotene from yellow vegetables • Vit A exists in 3 forms: alcohol (retinol), aldehyde and retinoic acid • Retinol and retinoic acid have roles as protein receptors • Rentinal (aldehyde) is a light-sensitive compound with a role in vision Prentice Hall c2002 Chapter 7 70 Fig 7.27 Formation of vitamin A from b-carotene Prentice Hall c2002 Chapter 7 71 B. Vitamin D • A group of related lipids involved in control of Ca2+ utilization in humans • Fig 7.28 Vitamin D3 and 1,25-dihydroxycholecalciferol Prentice Hall c2002 Chapter 7 72 C. Vitamin E (a-tocopherol) • A reducing reagent that scavenges oxygen and free radicals • May prevent damage to fatty acids in membranes Fig 7.29 Vitamin E (a-tocopherol) Prentice Hall c2002 Chapter 7 73 D. Vitamin K (phylloquinone) • Required for synthesis of blood coagulation proteins • A coenzyme for mammalian carboxylases that convert glutamate to g-carboxyglutamate residues • Calcium binds to the g-carboxyGlu residues of these coagulation proteins which adhere to platelet surfaces • Vitamin K analogs (used as competitive inhibitors to prevent regeneration of dihydrovitamin K) are given to individuals who suffer excessive blood clotting Prentice Hall c2002 Chapter 7 74 Structure of vitamin K and Vit K-dependent carboxylation Prentice Hall c2002 Chapter 7 75 7.14 Ubiquinone (Coenzyme Q) • Found in respiring organisms and photosynthetic bacteria • Transports electrons between membraneembedded complexes • Plastoquinone (ubiquinone analog) functions in photosynthetic electron transport Prentice Hall c2002 Chapter 7 76 Fig 7.30 (a) Ubiquinone, (b) Plastoquinone • Hydrophobic tail of each is composed of 6 to 10 five-carbon isoprenoid units • The isoprenoid chain allows these quinones to dissolve in lipid membranes Prentice Hall c2002 Chapter 7 77 Fig 7.31 • Three oxidation states of ubiquinone • Ubiquinone is reduced in two one-electron steps via a semiquinone free radical intermediate. Reactive center is shown in red. Prentice Hall c2002 Chapter 7 78 7.15 Protein Coenzymes • Protein coenzymes (group-transfer proteins) contain a functional group as part of a protein or as a prosthetic group • Participate in: (1) Group-transfer reactions (2) Oxidation-reduction reactions where transferred group is a hydrogen or an electron • Metal ions, iron-sulfur clusters and heme groups are commonly found in these proteins Prentice Hall c2002 Chapter 7 79 Fig 7.32 Stereo view of oxidized thioredoxin • Cystine group is on the surface (sulfurs in yellow) Prentice Hall c2002 Chapter 7 80 7.16 Cytochromes • Heme-containing coenzymes whose Fe(III) undergoes reversible one-electron reduction • Cytochromes a,b and c have different visible absorption spectra and heme prosthetic groups • Electron transfer potential varies among different cytochromes due to the different protein environment of each prosthetic group Prentice Hall c2002 Chapter 7 81 Fig 7.33 (a) Heme group of cyt a Prentice Hall c2002 Chapter 7 82 Fig 7.33 (b) Heme group of cyt b Prentice Hall c2002 Chapter 7 83 Fig 7.33 (c) Heme group of cyt c Prentice Hall c2002 Chapter 7 84 Fig 7.34 Absorption spectra of oxidized and reduced cytochrome c • Reduced cyt c (blue) has 3 absorbance peaks: a,b,g • Oxidized cyt c (red) has only a g (Soret) band Prentice Hall c2002 Chapter 7 85