An Introduction to Enzyme and Coenzyme Chemistry, 3Rd Ed. TDH

An Introduction to Enzyme and Coenzyme Chemistry, 3rd Ed. T. D. H. Bugg, Wiley, New York, 2012

Hydrolytic Enzymes:

Proteases Chapter 5, pp 79-92 Phosphatases Chapter 5, pp 95-102

Cofactors (Coenzymes): Thiamin Chapter 7, pp 165-188 2 e– Pyradoxal phosphate Chapter 9, pp 197-212 Nicotinamide Chapter 6, pp 115-122 redox 1 e– or 2 e– Flavin Coenzymes Chapter 6, pp 122-133 Heme Chapter 6, pp 136-140 1 e– mutase Vitamin B12 Chapter 11, pp 225-229

139

Enzymes: all enzymes are proteins catalysts speed up reactions by lowering the activation energy (ΔG‡), NOT by changing the thermodynamics of the reaction (ΔG°)

Stabilization of the transition state Bringing reactants together in the proper orientation for the reaction to occur 140

70 Proximity Br Br O O O -1 -1 - K = 1 M s + CH3CO2 + rel H3C O H3C O CH3 -O

Br O - ArO- O -1 O Krel = 220 s effect molarity 220 M O

- O CO2

Br O O - - ArO 4 -1 4 Krel = 5 x 10 s effect molarity 5 x 10 M O O

-O2C O

Br O O - 6 -1 6 - ArO Krel = 2 x 10 s effect molarity 2 x 10 M O O

- CO2 O

Br O

O

-O2C

Br O O 7 -1 7 O O Krel = 1 x 10 s effect molarity 1 x 10 M - ArO- O - O CO2 O 141 Bugg, pp 30-32

Br O Br O O 4 -1 O Krel = 5 x 10 s -O2C - CO2

reactive conformation for cyclization reaction

Br O

O held (pre-organized) in 6 -1 the reactive conformation Krel = 2 x 10 s - CO2

Br O

O

-O2C

142

71 Mechanism:

Mechanisms can not be proven absolutely, they can only be disproved (inconsistent with the available data)

Accepted mechanism is based on the preponderance of the available evidence

• consistent with the products - labeling studies • intuition: consistent with well-known chemistry and widely accepted mechanistic tenets • consistent with modest changes in the substrate • kinetic rate expression • model reactions - easier to study

143

Enzyme Commission (EC) classification – IUBMB #.##.##.X##

Class Reaction EC 1 Oxidoreductases – calatyzes oxidations and reductions EC 2 Transferases – functional group transfer

EC 3 Hydrolases – hydrolysis (overall addition of H2O) to a substrate to give two products

EC 4 Lyases – non-hydrolytic addition of removal of groups (i.e., H2O, NH3, etc) from a substrate. EC 5 Isomerases (mutase) – product is a structural isomer of the substrate EC 6 Ligases – joins two substrates by a bond formation reaction of two substrates using ATP to drive the reaction.

EC#’s define a biochemical reaction, not a specific enzyme

http://www.brenda-enzymes.org/index.php4 144

72 Simple enzymes: consists only of the proteins. Complex Enzyme: Protein contains other non-protein group(s) that assistant in the catalysis = coenzyme (vitamins or metal ions)

Coenzymes are bound to the protein by non-covalent interactions Prosthetic group: cofactor is covalently bound to the protein

Holoenzyme – protein – coenzyme complex Apoenzyme – protein only, missing the coenzyme.

145

Rates, equilibria and reaction order Velocity: v = k [S] k1 S P k-1

v1 = k1[S] ____[P] ____ k1 Keq = = v-1 = k-1[P] [S] k-1

Reaction Order: S P first order reaction v = k [S]

SA + SB P second order reaction v = k [SA] [SB]

half-life t½ = ln2/k (k = first order rate constant)

Rate-limiting (determining) step: slowest step in a multi-step chemical reaction. The overall rate of a reaction is dependent on the rate-limiting step

146 Bugg, pp 52-59

73 Plot of substrate concentration versus reaction velocity

k1 k2 S + E [S • E] P + E k-1 k-2

Steady-state approximation: [S•E] is constant

Vmax : the maximum velocity at saturating [S] Km : [S] at half the Vmax; rough estimate of the affinity of the S kcat : Vmax/[E] ; turnover number Vmax/Km or kcat/Km : catalytic efficiency of the reaction

147

Proteases: catalyzes the hydrolysis of peptide bonds Bugg, Chapter 5, pp. 81-98

O R O R O O R O R O H H protease H H H3N N N H3N N + N N N N CO H O N O H3N N CO H H 2 2 H 2 H O R O R H O R O R

1. Serine protease Bugg, p. 84 2. Cysteine protease p. 89 3. Aspartyl protease p. 95 4. Zinc (metallo) protease p. 92

•chymotrypsin: cleaves at the C-terminal side of aromatic residues Phe, Tyr, Trp • trypsin: cleaves at the C-terminal side of basic residues Arg, Lys but not His

148

74 Serine Protease: Chymotrypsin oxy-anion hole NH HN O

R2 R1 N H Ser 195 O Ser195 O H H R1CO2H H2O His57 N + His57 N

N R2-NH2 N H H Asp CO - - 102 2 Asp102 CO2

149 Bugg, p. 82

Catalytic triad of α-chymotrypsin

His-57

Asp-102 Ser-195

pdb code: 5CHA 150

75 Active site of bovine trypsin with a bound inhibitor

Asp-102

Asp-189 His-57

Ser-195

O O OEt EtO OEt H H H H tBuO N N B tBuO N N B N OEt N O Ser-175 H H O O O O

NH3 NH3 pdb code: 1BTX Katz, B. A.; Finer-Moore, J.; Mortezaei, R.; Rich, D. H.; Stroud, R. M. Biochemistry 1995, 34, 8264-8280. 151 Bugg, p. 81

Oxy-anion hole of trypsin

Asp-194 Gly-193 Gly-196 Ser-195 Gln-192 Asp-194 Gly-193 Gly-196 Ser-195 His-57 Gln-192

Asp-102 Asp-102 His-57

O EtO OEt H H tBuO N N B N O Ser-175 H O O

NH3 pdb code: 1BTX 152

76 Cysteine Protease: Papain (212 amino acids) active-site cysteine and histidine, overall mechanism is similar to serine proteases usually not a digestive enzyme (intracellular) oxy-anion hole NH HN O Cys R2 25 S R1 N H H R1CO2H Cys 25 H2O + S + H His159 N R2-NH2 N His159 N H N H

153 Bugg, p. 85

Structure of papain with a bound inhibitor

Gln19 Asp158

His157 oxyanion hole

Cys25

O O H H O O papain N H S Cys25 N O N N H H H O N N O O N H H H O O H oxyanion O N pdb code: 1BP4 H hole 154 Asn Gln1919

77 Aspartyl Protease Mechanism: Renin, HIV protease Bell shaped pH vs. rate profile: max rate at pH ~ 2.4 indicative of general acid-general base catalysis. Asp Asp Asp

O O Asp O _ HO O H H O _ O N O O R H O O H R' N H R H HO R'

155 Bugg, p 91

HIV Protease: an Aspartyl Protease

Catalytic Asp-25 with a bound inhibitor

Ph O O OH O H CH3 N N N N N

CH3 H O H O Ph pdb code: 1HVJ inhibitor 156

78 Interaction of the catalytic Asp-25 with the bound inhibitor

Ph O O OH O H CH3 N N N N N

CH3 H O H O Ph pdb code: 1HVJ inhibitor 157

Metallo-protease (zinc): carboxypeptidase: important catalytic groups: Glu-270, Tyr-248 Zn ion coordination: Glu-72, His-196, His-69 General base mechanism

Nucleophilic mechanism (acyl enzyme complex)

158 Bugg, p. 88

79 Active site of carboxypeptidase

Arg-145

Tyr-248 Bound His-69 ligand

Zn Glu-72 H2O His-196 Glu-270

pdb code: 2CTC 159

Phosphate ester hydrolysis

1. Phosphatases: over transfer (hydrolysis) of a phosphate monoester to water 2. Phosphodiesterases: hydrolysis of a phosphodiester to a phosphate monoester and an alcohol 3. Kinases: transfer of the γ-phosphate group of ATP to an acceptor group Glucose 6-phosphatase -2 O3PO + H2O HO

HO HO -3 HO HO OH + PO4 OH - H2O HO HO (Pi) glucose-6-phosphate Fructose 1,6-bisphosphatase: M+2 dependent

-2 -2 O3PO OH + H2O O3PO O OH O + PO -3 HO HO 4 (P ) -2 - H2O i CH2OPO3 CH2OH HO HO fructose-1,6- bisphosphate Alkaline phosphatase: M+2 dependent 160 Bugg, pp. 95-102

80 Phosphatases: General acid-base mechanism

161

Phosphatases: Covalent mechanism

SN2 mechanism

162

81 Glucose 6-phosphatase: covalent catalysis

Fructose 1,6-bisphosphatase: General acid-base catalysis

163

Associative vs dissociative mechanism: Chiral phosphate Use three isotopes of oxygen, 16O, 17O, 18O

_ O O +R OH O 2 _ P OR _ R OH R2O P _ 2 R1O P 1 + _ -or- O _ _ O O O O O

_ S _ S + H O S 2 O P _ _ P O RO P _ ROH + -or- _ O O _ _ O O O O

Does the observation of stereochemically defined products (retention or inversion of stereochemistry) rule out the dissociative mechanism involving metaphosphate ion?

164

82 Tyrosine phosphatase conserved aspartate, cysteine, and arginine Asp O O H O _ Tyr O P _ S Csy + H2O -3 _ O Tyr OH + PO4 O (Pi) H + H N H N H HN Arg

165

Phosphodiesterase: Ribonuclease (RNase): catalyzes the hydrolysis of the phosphodiester bond of RNA. Catalytic groups are His-12 and His-119 B His B O O His O O N NH OH N NH RNase O O O H O B P O- O B - P O HO O - O O O H OH His OH N O H O His N N H N H

Bell shaped pH vs. rate profile: max rate at pH ~ 6.0 indicative of general acid-general base catalysis. Mechanism requires both an imidazole and imdiazolium for catalysis

pKa3 of hisidine is ~ 6.1 166 Bugg, pp. 99-100

83 Some Cofactors O NH2 OH H O N S O P OH O O HO N P OH P OH N N O O N + HO H HO O N N OH O N O S N O P O OH NH2 N Pyridoxal Phophates Thiamin Diphosphate H Biotin HO P O OH O O OH H H N N N NH2 O P O SH N OH O O OH OH O OH OH N - Coenzyme A (CoA) O O O N O O O O O N N P N N P P O NH2 O- - - O O OH O N OH O NH2 OH O N Na+ Na+ HO HN O HN FMN O H2N O FAD N NH2 O N C N NH Co O 2 O H NH N N 2 N N H2N N NH2 O O N N N Fe N O O P O P O O N O O N OH OH N NH HO OH HO O N O H R= H NADH R HO HO H R= -PO H NADPH O O 3 2 O OH -O P Heme O O O H H NH OH OH N NH2 NH2 O H Vitamin B12 O O N HO N O NH N N O NH O O H OH H HS HO Folic Acid OH O Glutathione S S Lipoic Acid 167

O Thiamin Dependent Reactions (Vitamin B1) O- S O O P P O- (Bugg, Chapter 7, pp. 165-8) N N + -O O

N Pyruvate Decarboxylase NH2

O thiamin O Thiamin Diphosphate (pyrophosphate) H3C C CO2H H3C C H + CO2 Acetolactate synthase

O thiamin O OH 2 + CO2 H3C C CO2H H3C C C CO2H CH3

Pyruvate dehydrogenase: pyruvate decarboxylase, dihydrolipoyl transacetylase, dihydrolipoyl dehydrogenase, O + thiamin + lipoic acid + CoA-SH H3C C CO2H Acetyl-CoA + CO2 pyruvate (CH3COS-CoA) O O - O O thiamin, CO2 CoA-SH Lys H3C S Lys N N S-CoA H H S S - CO2 O SH 168

84 Mechanistic insight into thiamin dependent reactions: Breslow, R. J. Am. Chem. Soc. 1958, 80, 3719.

O - O - S O O D O O P S O O P H D O P O- P O- 2 N N - O N N - O + O + O t ~ 20 min 1/2 N N NH2 NH2

pKa measured to be between 12-18

Benzoin condensation O 2 PhCHO + - CN Ph + - CN Ph pKa ~ 9.3 OH

169

Mechanism of pyruvate decarboxylase:

S PPO H O N + H3C C CO2H

R

Mechanism of acetolactate synthase:

170

85 Pyruvate Dehydrogenase: dihydrolipoyl transacetylase

171

Transketolase: carbohydrate biosynthesis

H C OH H C OH 2 CHO 2 O O H OH CHO H OH H OH thiamin-PP HO H + H OH + H OH H OH H OH H2C OPO3 H2C OPO3 H2C OPO3 H OH H OH D-ribulose-5-P glyceraldehyde-3-P D-ribose-5-P H2C OPO3 (C5 ketose) (C3 aldose) (C5 aldose) sedohepulose-7-P (C7 ketose)

172

86 Thiamin acidity and anion stability is special to the N-alkyl thiazole ring system: anion is stabilized by three major resonance strictures: two are ylides, one is a carbene

R N S R N S R N S

ylide carbene ylide central carbon has 6 electrons

Nature may have tried the imidazole and oxazole ring systems, but these would not have performed the necessary chemistry.

H H R N N R N O

Not acidic more acidic than thiamin, but the anion is hydrolytically labile 173

Pyridoxal Phosphate (PLP) dependent enzymes (Vitamin B6) (Bugg, Chapter 9, pp 197-212)

O H NH2 OH

2- OH 2- OH 2- OH O3PO O3PO O3PO N N N

pyridoxal pyridoxamine pyridoxine

Involved in amino acid biosynthesis, metabolism and catabolism racemase, CO - H β H epimerase 2 - CO - R H R' CO2 R α 2 NH3 NH3 NH3

reaction at the L-amino acid D-amino acid β or γ carbon decarboxylase

H transaminase - R H CO2 R NH3 NH2 H2O CO - R 2 O 174

87 Pyridoxal is often covalently bound to the resting enzyme as a Schiff base to the sidechain of an active site lysine residue.

Lys N H O PO

N H

175

Decarboxylase: L-DOPA decarboxylase HO CO2 HO NH3 NH HO 3 HO Dopamine CO2

HO NH 3 HO NH3

N N H H Serotonin

176

88 Transaminase: amino acid biosynthesis

177

Stereochemistry of the transamination reaction: proton transfer is mediated by an active site lysine

- - R CO2 R CO2 H H H NH N H N H 2 H H H - OH R CO2 O O PO + PO PO O N N N H H H

Enzyme Enzyme Enzyme

H2N H N H2N OP 2 OP H OP H H H H NH R H NH NH R R N N N H O -O C H O H O -O2C 2 -O2C

178

89 Stereochemistry of transamination (aspartate aminotransferase)

Lys-258

Arg-266

Arg-386 Arg-292

Enzyme Enzyme Enzyme H H H N N Arg N Arg Arg H2N H N H2N H N NH2 2 NH2 H 2 - H2N NH2 H2N - N OPO3 H - H OPO3 Arg NH N OPO3 N 2 H Arg H N H Arg NH2 NH2 H 2 - H H N H O2C H NH H2N - 2 - NH O2C NH O2C N N - H O N - H O O2C - H O O2C O2C NH 2 NH2 NH2 H2N Arg H N Arg H2N Arg N 2 N N H H H 179 Pdb code: 1AJS

Reactions at the β- and γ-carbons of amino acids

OH threonine β H dehydratase CO - CO - 2 + NH H3C α 2 H3C 4 NH3 O

Threonine

H serine hydroxymethyl - " H " HO CO2 transferase CO - 2 + O NH 3 H NH3 formaldehyde equivalent tryptophan H - synthase H HO CO2 CO - + 2 + H2O N NH3 NH H N 3 H

methionine β H lyase, H3CS - CO2 H O CO - α 2 2 + NH + CH SH γ H3C 4 3 NH3 O

180

90 Threonine Dehydratase

181

Tryptophan Synthase: reactivity of indole toward electrophiles:

Similar to the mechanism of threonine dehyratase an electophilic PAL-amino acid complex is generated

Enzyme

B H OH Enzyme - CO2 :B H N H O PO

N H 182

91 Tryptophan Synthase (con’t):

183

Serine hydroxymethyl transferase: Bugg, Ch. 9, pp. 206-7 one-carbon (methyl) donors in biology

H H H2N N N H CO - C - PAL, Folic acid 2 HO CO2 HN N NH HN 3 2 NADPH - O H2C N CO2 O methylene tetrahydrofolate

H2N N N - H CO2 H N N N 2 CO - HN 2 NADPH 2 N HN HN N O HN CO - HN 2 H - O O HN CO2 O Folic acid

CO - H2 NH H3N O O O N 2 H N NH H3CS - + -O P O P O P O 2 CO2 O N N O- O- O- S NH N O N N 3 -3 -4 H C - PO4 , - P2O7 3 HO OH N ATP HO OH

S-adenosyl methionine (SAM) 184

92 Methyl groups from: SAM Methylene tetrahydrofolate

O

H3C CH3 OH OH H3C HO N CH O OH O OH 3 H3C CH3 H3C H3C O O OH H3C O O O O CH O 3 CH3O O OH O NH CH3 2-O PO N CH3 O 3 O H3C O H C OH NH 3 OH 2 OCH3 HO Erythromycin Daunomycin

CH O O 3

NH H3C N N CH3 CH3

Physostigmine

185

Mechanism of serine hydroxymethyl transferase:

Note: this mechanism is not the same as on page 207 of Bugg, which is probably incorrect 186

93 Mechanism of methionine γ-lyase

Enzyme - H3CS CO2 :B H N H O PO

N H

187

Redox Cofactors

1.0 + - 0.82 O2 + 4H + 4e → 2 H2O

+3 - +2 0.40 cytochrome a3 (Fe ) + e → cytochrome a3 (Fe ) - 0.30 O2 + 2H2O + 4e → 2 H2O2 0.24 cytochrome c (Fe+3) + e- → cytochrome c (Fe+2) Eo' (volts vs SHE) 0 + - -0.20 FAD + 2H + 2e → FADH2 -0.32 NAD(P)+ + H+ + 2e- → NAD(P)H

-1.0

Bugg, Chapter 6 188

94