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
mutase 1 e– Vitamin B Chapter 11, pp 225-229
12

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
+ CH3CO2- +
Krel = 1 M-1 s-1
H3C O H3C O CH3 -O

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

CO2- O

Br O
O - ArO-
Krel = 5 x 104 s-1 effect molarity 5 x 104 M
O O

-O2C O

Br O
O - ArO- Krel = 2 x 106 s-1 effect molarity 2 x 106 M
O O

CO2- O

Br
O

-O2C

Br
O O O O Krel = 1 x 107 s-1 effect molarity 1 x 107 M
- ArO-
O O
CO2-
O
141

Bugg, pp 30-32

Br
O Br
O
O
O Krel = 5 x 104 s-1
-O2C
CO2-

reactive conformation for

cyclization reaction

Br
O
held (pre-organized) in
O
the reactive conformation Krel = 2 x 106 s-1
CO2-

Br
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

144

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

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

Reaction Order:
S P first order reaction v = k [S]
SA + S B 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 protease O R O R O
H H H H
H3N N N H3N N + H N N
N N N CO2 H2O N O 3 N CO2
H H H
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
R
R1 N 2
H Ser195
Ser195 O
O H
H R1CO2H
H2O

His57
His57 N +
N
N R2-NH2
N H
H Asp102 CO2-
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 EtO OEt
O OEt H H
H H tBuO N N B
tBuO N N B N O Ser-175
N OEt 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
R2 Cys25
R1 N S
H H R1CO2H
Cys25
S
H2O + +
H His159 N
R2-NH2
His159 N
N
H
N
H

153

Bugg, p. 85

Structure of papain with a bound inhibitor

Gln19
Asp158

His157

oxyanion

hole

Cys25

O O
O O papain H H
H N S Cys25
N H O N N
O N N H H
O O N
H H H
O O
H oxyanion

O N
pdb code: 1BP4
H hole
154

Asn19
Gln 19!

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
_
H H O_ HO 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

ligand


His-69

Zn
Glu-72

H 2O

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
HO HO OH + PO4 -3
OH - H2O
HO HO (Pi)
glucose-6-phosphate

Fructose 1,6-bisphosphatase: M+2 dependent

-2 -2
O3PO OH + H2O O3PO
O OH
O + PO4 -3
HO HO
-2 - H2O (Pi)
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
O +R2OH _
R2O P _ P OR2
R1O P _ R1OH + _ -or- O _
_ O O O
O O

_ S S _
S + H2O
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
_ O Tyr OH + PO4 -3
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
O His N NH
O
OH
N NH O
RNase
O O H O
P O- B
O B -O
P HO O
-O O O
H OH
His N O
H OH
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
S OH H O N
O O P OH
HO
O N
P OH P OH
N N O O O N
+ HO H HO N
N OH O
N O S N O P O
NH2 N OH
Thiamin Diphosphate Pyridoxal Phophates
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
OH OH N O - 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
O N OH HO
Na+ Na+ HN O
HN O FMN H2N
O FAD N NH2
O N C N
NH2 Co
O O H N N
NH2
N N
N H2N NH2
O O N N
N N Fe N O
O O P O P O O O
N
OH OH N
NH
HO OH HO O O N
HO H
R= H NADH R HO H
R= -PO3H2 NADPH O O
OH -O O
P
O Heme O
O H H
NH OH
OH N NH2 NH2 O
H Vitamin B12
O O N HO N O
NH N N
H OH O
NH O O H
HO HS OH
Folic Acid
O Glutathione S S
Lipoic Acid
167

Thiamin Dependent Reactions (Vitamin B1)

S
O
O
-
O O P
(Bugg, Chapter 7, pp. 165-8)
N N
P
O
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 H3C C CO2H H3C C C CO2H + CO2
CH3

Pyruvate dehydrogenase: pyruvate decarboxylase,

dihydrolipoyl transacetylase, dihydrolipoyl dehydrogenase,

O
H3C C CO2H + thiamin + lipoic acid + CoA-SH Acetyl-CoA + CO2
pyruvate (CH3COS-CoA)
O
O thiamin, CO2- O CoA-SH O
Lys H3C S Lys
N N S-CoA
S S H - CO2 O SH H

168

84

Mechanistic insight into thiamin dependent reactions:


Breslow, R. J. Am. Chem. Soc. 1958, 80, 3719.

O
O S O-
S O- D O O P
O O P P
H
P D2O O-
O- N N -
O O
N N -
O O +
+
t1/2 ~ 20 min 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:

PPO S O
H
N + H3C C CO2H

Mechanism of acetolactate synthase:

170

85

Pyruvate Dehydrogenase: dihydrolipoyl transacetylase

171

Transketolase: carbohydrate biosynthesis

H2C OH H2C OH
CHO
O H OH O
thiamin-PP CHO
H OH H OH 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
(C5 ketose) D-ribose-5-P H2C OPO3
(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-O OH 2-O PO OH 2-O OH

3PO 3 3PO

N N N

pyridoxal pyridoxamine pyridoxine

Involved in amino acid biosynthesis, metabolism and catabolism

racemase,
β H epimerase CO2-
H H
CO2- R CO2- R
R' α
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 CO2-
R
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

NH3
HO HO
Dopamine
CO2

HO NH3 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 N H N
NH2 H H
H H
OH R CO2- O O
PO + PO PO
O
N N N
H H H

Enzyme Enzyme Enzyme

H2N H2N H2N

OP OP
H OP
H H
H H NH
R H NH NH R
R N
N N H O
-O2C H O H O -O2C
-O2C

178

89

 Stereochemistry of transamination (aspartate aminotransferase)

Lys-258

Arg-266

Arg-386

Arg-292

Enzyme Enzyme Enzyme
H H H
N Arg N Arg N Arg
H2N H2N H2N
H H2N NH2 NH2 H2N NH2
H2N OPO3-
N OPO3- H OPO3-
H
Arg NH2 N H N
H Arg NH2 Arg NH2 H
H2N -O C
NH H2N -O C H H2N - H NH
2 H 2 NH O2C
N N
-O C H O N -O H O
2 -O C H O 2C
2
NH2 NH2 NH2
H2N Arg H2N Arg H2N Arg
N N N
H H H
179

Pdb code: 1AJS

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

OH threonine
β H dehydratase CO2-
H3C CO2- H3C + NH4
α
NH3 O

Threonine

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

methionine
β H lyase,
H3CS CO2- H2O CO2-
γ αNH H3C + NH4 + CH3SH
3
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 CO2-
C CO2- PAL, Folic acid
HO HN
N HN
NH3 2 NADPH O H2C N CO2-
O

methylene tetrahydrofolate
H2N N N H
CO2- H2N N N
HN CO2-
N HN
2 NADPH HN
N HN
O HN CO2- H
O O HN CO2-
O
Folic acid

CO2-
H
NH2 H3N
O O O N
H N NH2
H3CS CO2- + -O P O P O P O O N N
O- O- O- N
S O N
NH3 N
- PO4-3, - P2O7-4 H3C
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 CH3 O OH O
H3C OH CH3
H3C
H3C O O OH H3C O
O
O O O CH3 CH3O O OH O NH
CH3 2-O N
CH3 3PO O
H3C O
O
H3C OH NH2
OCH3 OH
HO

Erythromycin Daunomycin

CH3
O O

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

0.40 cytochrome a3 (Fe+3) + e- → cytochrome a3 (Fe+2)

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

-1.0

Bugg, Chapter 6

188

94