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The Organic Chemistry of

Enzyme-Catalyzed Reactions
Chapter 3
Reduction and Oxidation

Redox Without a Coenzyme


Internal redox reaction

Reaction Catalyzed by Glyoxalase


Scheme 3.1

O O

OH

CH3C CH

CH3 CHCOOH

3.1
methylglyoxal

3.2
lactic acid

Looks like a Cannizzaro reaction

Cannizzaro Reaction Mechanism


O

Ph C H +

Ph C H

OH

Ph COO
oxidized

HO
O
O
Ph C

Ph C H
H

HO

Scheme 3.2

Ph CH2OH
reduced

Reactions Catalyzed by Glyoxalase I


and Glyoxalase II
reduced
oxidized

glutathione
O O
CH3 C C H

+GSH

glyoxalaseI

3.3

HO

CH3 C

SG

H 3.4

Scheme 3.3

+H2O

HO

CH3 C

glyoxalaseII

SG

3.4
OH

CH3 CHCOO

+GSH

Glutathione (GSH)
COO

CH2SH

H3N CHCH2CH2 CNH CH C NHCH2CO2


(

Hydride Mechanism for Glyoxalase


H B

B
O O
CH3

C C H

H SG

CH3

oxidized

reduced

C SG

OH O
CH3

C
H

H2O

OH
GSH+

C SG

CH3 CH

COO

Scheme 3.4
Intramolecular Cannizzaro reaction

glyoxalaseII

Evidence for a hydride mechanism - when run


in 3H2O, lactate contains less than 4% tritium
NMR experiment provided evidence for a
proton transfer mechanism:
Enzyme reaction followed by NMR
At 25 C in 2H2O, 15% deuterium was
incorporated
At 35 C, 22% deuterium was incorporated

Enediol Mechanism for Glyoxalase


B
CH3

+GSH

CH3

B:

H
O

OH

C
H

Scheme 3.5

B:

SG

CH3

cis-enediol

H
HO

B+ H

SG

3.5

noexchange
withsolvent

CH3

HO

SG

Reaction of Glyoxalase with


Fluoromethylglyoxal
Another test for the mechanism
O O
FCH2C CH

glyoxylase

3.6

Scheme 3.6

GSH

FCH2

HO

SG

CH3C

C
3.8

3.7

same oxidation state

SG

Hydride Mechanism for the Reaction of


Glyoxalase with Fluoromethylglyoxal
O O
FCH2C CH

GSH

B+ H
O

FCH2C

3.6

Scheme 3.7

HO O
F
SG

HO

CH2 C C SG
H
B:

3.7

CH2

O
CSG

CH3C

C
3.8

SG

Enediol Mechanism for the Reaction of


Glyoxalase with Fluoromethylglyoxal
B+ H
O

FCH2C

O O
FCH2C CH

GSH

3.6

HO
SG

F CH2 C

O
F

C SG

HO

CH2

H
B+ H

B:

B+

SG

a
O

CH3C

C
3.8

Scheme 3.8

SG

CH2

HO

SG

FCH2

HO

H
3.7

SG

Hydride Mechanism for the Reaction of Glyoxalase


with Deuterated Fluoromethylglyoxal
B+
O O
FCH2C CD

GSH

3.9

H
O

FCH2C

HO
SG

CH2

O
C

SG

CH3C

D
B:

Scheme 3.9

deuterium
isotope effect

F- loss
decreased

SG

Enediol Mechanism for the Reaction of Glyoxalase


with Deuterated Fluoromethylglyoxal
B+

H
O

FCH2C

O O
FCH2C CD

GSH

3.9

HO
SG

F CH2 C

C SG

HO

CH2

B+ D

B:

B+

CH3C

SG

CH2

HO

SG

F
O

SG

FCH2

HO

SG

Scheme 3.10

F- loss
increased

deuterium
isotope effect

Table 3.1. Comparison of Fluoride Ion Elimination with Fluoromethyl Glyoxal and [1-2H]Fluoromethyl
Glyoxal
Source

% Fluoride ion elimination


O

FCH

CH

FCH

CD

yeas t

32 .20.2

40 .70.2

rat

7.70.1

13 .30.9

mou se

26 .41.0

34 .80.5

yeas t/D2 O

33 .80.2

39 .10.4

increased F- loss
supports enediol
mechanism

Redox Reactions that Require Coenzymes


Nicotinamide Coenzymes (Pyridine Nucleotides)
Pyridine nucleotide coenzymes include
nicotinamide adenine dinucleotide (NAD+,
3.10a), nicotinamide adenine dinucleotide
phosphate (NADP+, 3.10b), and reduced
nicotinamide adenine dinucleotide phosphate
(NADPH, 3.11b)

NAD(P)+

NH2

NH2

O
N

CH2
OR' HO
O

O O
OP OP O CH2
O

O
HO

NH2
O

NAD(P)H

CH2

a,R'=H
b,R'=PO3=

3.10

O
NH2

OR' HO
O

OH

O O
OP OP O CH2
O

HO

OH

3.11

Enzyme without coenzyme bound - apoenzyme


Enzyme with coenzyme bound - holoenzyme
apoenzyme

coenzyme

holoenzyme

Called
reconstitution

Abbreviated Forms
O

H O

NH2

NH2

3.12

3.13

NAD(P)+
(oxidized)

NAD(P)H
(reduced)

Coenzymes typically derived from vitamins


(compounds essential to our health, but not
biosynthesized)
Pyridine nucleotide coenzymes derived from
nicotinic acid (vitamin B3, also known as
niacin)

Biosynthesis of Nicotinamide Adenine


Dinucleotide (NAD+)
COOH
=
O3PO

COOH
+
N
3.14

nicotinic acid
(vitamin B3)
niacin

PPi
O
OP2O63
OH OH
3.15

=
O3PO

ATP

NH2
N

PPi N
N

OH

O
OH

OH

CH2
OH HO
O

3.16

from ATP

O O
OP OP O CH2
O
O O
3.17

HO

OH

Gln
ATP

NH2

N
N

O O
CH2 OP OP O CH2
O
O O
OH HO
O
3.18

HO

NH2
N

OH

Scheme 3.11

Reactions Catalyzed by Pyridine


Nucleotide-containing Enzymes
H
C

OH

Oxidation
potential
NAD+/NADH
is -0.32 V

H
C

+NH3

Figure 3.1

Reactions Catalyzed by Alcohol


Dehydrogenases
Mechanism
B:

H
R C

R C

O H

+B

H O

H
NH2

NH2

R
3

In H2O, no 3H in NAD(P)H
Hydride mechanism

Scheme 3.12

Reaction Catalyzed by Alcohol


Dehydrogenases Using Labeled Alcohol
H

O
NH2

* 2OH +
RCH
N
R

Scheme 3.13

H2O

O
R

*H

*H O
NH2

+
N
R

No *H found in H2O

Supports hydride mechanism

Test for a radical intermediate

Cyclopropylcarbinyl Radical Rearrangement


k=108s1

3.19

Scheme 3.14

3.20

Test for the Formation of a Radical


Intermediate with Lactate Dehydrogenase
O

OH
CO2H

3.21

pigheart
lactate
dehydrogenase
NADH

Scheme 3.15
No ring cleavage - evidence
against radical mechanism

CO2H

Chemical Model for the Potential Formation of a


Cyclopropylcarbinyl Radical during the Lactate
Dehydrogenase-catalyzed Reaction
O
CO2Me

Scheme 3.16

AIBN

OSnBu3

OSnBu3
CO2Me

CO2Me

O
CO2Me

Should have seen ring opening in the enzyme


reaction if a cyclopropylcarbinyl radical formed

Nonenzymatic Reduction of -Chloroacetophenone


Another test for a radical intermediate
Nonenzymatic reaction
O

NADH
Ph

CH2Cl
3.23

Scheme 3.18

Ph
3.24

CH3

radical reduction
product

Horse Liver Alcohol Dehydrogenase-Catalyzed


Reduction of -Haloacetophenones
O

OH

HLADH
Ph

CH2X

Scheme 3.19

NADH

Ph

*
3.25

hydride reduction product


(stereospecific)
X = F, Cl, Br

Supports no radical intermediate


When X = I, get mixture of 3.25 (X = I) +
Electron transfer is possible
if the reduction potential is
low enough

Ph

O
CH3

(radical reduction
product)

Stereochemistry
An atom is prochiral if by changing one of its
substituents, it changes from achiral to chiral

Stereochemistry:
Determination of the chirality of an isomer of alanine
R,S Nomenclature
H3C
H3N

COO

Figure 3.2

D
B

lowestprioritybehind
counterclockwise
(S)

Determination of Prochirality
Caacd
prochiral

Cabcd
chiral

proRhydrogen
H

2H

CH3

OH

CH3

OH

prochiral

chiral

proShydrogen
H
CH3

Figure 3.3

H
OH

prochiral

2H

CH3

OH
S

chiral

Determination of sp2 Carbon Chirality


Determine the priorities of the three
substituents attached to the sp2 carbon
according to the R,S rules
If the priority sequence is clockwise looking
down from top, then the top is the re face; if it
is counterclockwise, then it is the si face

Determination of Carbonyl and


Alkene (sp2) Chirality
siface

siface

CH2

O
CH3

reface

Figure 3.4

CH3

reface

Reaction of Yeast Alcohol Dehydrogenase


(YADH) with (A) [1,1-2H2]ethanol and NAD+
and (B) Ethanol and [4-2H]NAD+
H

O
NH2

CH3CD2OH

YADH

O
NH2

+CH3CDO

3.26
D

H
NH2

B
N
R

Scheme 3.20

CH3CH2OH

YADH

O
NH2

N
R
3.27

+CH3CHO

Reaction of YADH with (A) [4-2H]NAD2H Prepared in Scheme


3.20A; (B) Reaction of YADH with [4-2H]NAD2H Prepared in
Scheme 3.20B; (C) Reaction of YADH with 3.28 and NAD+
D

No 2H

NH2

YADH

+CH3CHO

No H

O
NH2

3.26

3.27

H
+

Scheme 3.21

O
NH2

N
R

YADH

CH3COH
3.28

3.28

YADH

+CH3CHO

NH2

NH2

R
3.26
H

stereospecific

CH3CHO

CH3CH2OH

only one H is transferred


HR O

HS

re-face
R N

NH2
N

HS
O
H2N

R
3.29

HR

Not all enzymes transfer the same hydride


(A) Reaction of YADH with [1,1-2H2]ethanol and NAD+;
(B) Reaction of glyceraldehyde-3-phosphate
dehydrogenase (G3PDH) with the cofactor produced in A
and glycerate 1,3-diphosphate
H
A

CH3CD2OH +NAD+

YADH

O
NH2

pro-R
+CH3CDO

N
R
3.26
D
O
B

3.26 + H2C

CH C OP

OP OH

Scheme 3.22

G3PDH

O
NH2

+N
R

pro-S transferred

H2C

CH CHO

OP OH
3.30

+Pi

Transition State for Hydride Transfer


Anti- and syn- conformations of NADH
Figure 3.5

HS O

HR
NH2

RO

RO

anticonformation

HS

H2N

H
OH OH

HR

syn-axial
electrons
assist

N
O
OH OH

Boat-like TS

synconformation
proS
transfer

proR
transfer

The enzyme may drive equilibrium


Boat-boat equilibria of NADH
antiNADH

HR

HS
HR

CONH2

CONH2

HS

RO
O

HO

RO
O

OH

OH

HO

HRtransfer

N
O

Figure 3.6

HO

HS

H2NOC

HR
H2NOC
RO

HR

synNADH

HS

RO
O

OH

HO

OH

HStransfer

Oxidation of Amino Acids to Keto Acids


Possible mechanism for the reaction catalyzed
by glutamate dehydrogenase
K125

NH
.. 2

Hydride transfer

CONH2
+N

OOC

H
+
NH2

K125

D165
H

OH

CONH2

CO2
H3N K113

CO2

NH
.. 2

CO2

N
R

H3N K89

+
H OOC D165
NH2

COO

H3N K113

H3N K89
K125
K125

NH3

NADPH

HOOC
O

NH3
CO2

CO2

Scheme 3.24

D165

H3N K113

H3N K89

NH3
H
O

+ OOC
NH3

D165

CO2
CO2

H3N K113
H3N K89

Oxidation of Aldehydes to Carboxylic Acids


(A) Covalent catalytic mechanism for the oxidation of
aldehydes by aldehyde dehydrogenases; (B) noncovalent
catalytic mechanism for the oxidation of aldehydes by
aldehyde dehydrogenases
+

B:
O

S
H

covalent catalysis

Scheme 3.25

S
3.32

NAD+

O H

Hydride transfers
H

RCHO+H2O

3.31

B:

+NADH

O
R

OH
H

3.33

R
NAD+

via hydrate

OH

+NADH

O
R

OH

Oxidation of Deoxypurines to Purines


Mechanism for the oxidation of inosine 5-monophosphate
by inosine 5-monophosphate dehydrogenase
O

H B+
N

HN
N
X H 3.36
B:

HN

RP

RP

H O

NH2

inosine MP

NH2

N+

O
X H
B:

N
H

B:
N

HN

Scheme 3.27

H B+

H OH HN

RP

:B

O
X

xanthine MP

B+

:B
N

HN

RP

3.37

N
H
B+

N
RP

An Atypical Use of NAD+


Reaction catalyzed by urocanase
NAD+ in a Nonredox Reaction
H
N

H
N

COOH
urocanase
D2O

N
3.39

Scheme 3.28

D
COOH
D

OH
3.40

Urocanase Reaction Run with a [13C]


Pseudo-substrate
apo-urocanase reconstituted
with [13C]NAD+
O

substrate
13

H
N

NH2

COOH
N+

N
H

13

3.41

exchangeable
proton

reduced
sidechain

R
3.42

Adduct Isolated after Chemical Oxidation


H
N

13

COO

N
O

13

NH2
N
R
3.43

NMR determined

Mechanism Proposed for Urocanase


H

N+

COO

H
N

:B
N

exchangeable

OH
+NAD+

N
R

COO

+N

COO

N+

NH2

H
COO

OH
N
R

oxidativequenchoxidizes
thisreducedadduct

NH2

N+

When3.41isused,
thereactionstopshere.

COO
H

NH2

H
N

COO
H
O

solvent incorporated

OH

O
NH2

H
H

H
B:

N
R

B+

O
NH2

Scheme 3.29

Flavin Coenzymes
Biosynthetic conversion of riboflavin
to FMN and FAD
NH2
O
CH2O

CH2OH
(CHOH)3

8a
8
7

N 10a N
10

3.48

CH2O

O ATP
NH

riboflavin
(vitamin B2)

Scheme 3.31

ADP

O ATP

PPi

FMN

FAD

O CH2

O
NH

N
3.50

HO

NH

N
3.49

CH2
N

(CHOH)3

CH2

CH2

N 4a
5

(CHOH)3

OH

N
N

Interconversion of the Three Oxidation


States of Flavins
oxidized

semiquinone

reduced

R
N

+1e
1e

NH

N
O

(Fl)

O
NH

N
3.51

some covalently attached to


The protein at these positions

1e

N
N

N
O

Fl

Scheme 3.32

O
NH

R
O

N
H
3.52
FlH

R
N

+1e

N
O

O
H

Redox Reactions Catalyzed by Flavindependent Enzymes


H
C

OH

H
C

NH2

CH2

CH2

CH

O
NADH

HS

SH

Figure 3.8

NH4+

CH

C
O

NAD+

Oxidases vs. Dehydrogenases


Mechanisms for an oxidase-catalyzed oxidation of
reduced flavin to oxidized flavin
R

N
N
H
B H

only if spin
inversion
occurs

O
NH

3.54

H2O2

NH

3.53
H2O2

O O

c
d

NH
O

Flox

H O O
OH

radical
combination

N
H

O O

e transfer

R
N

O O

Scheme 3.33

2ndetransfer
+H+
H

Oxidases use O2 for reoxidation


of reduced flavin coenzyme

Dehydrogenases Use Electron Transfer


Proteins to Reoxidize Reduced Flavin
Mechanism for a dehydrogenase-catalyzed
oxidation of reduced flavin to oxidized flavin
R
N
N
H

R
N

NH
O

Acceptor

O
NH

Acceptor

Scheme 3.34

R
N

NH

O
Acceptor

Mechanisms for Flavoenzymes


Overall reaction of flavoenzymes
Substrate
EnzymeFlH

EnzymeFlox
+

Scheme 3.35

Acceptor
(O2)

EnzymeFlH

Oxidizedsubstrate
(product)

EnzymeFlox

Reducedacceptor
(H2O2)

Mechanisms for Flavin-dependent Enzymes


Three types of mechanisms:
a carbanion intermediate
a radical intermediate
a hydride intermediate
Each of these mechanisms may be
applicable to different flavoenzymes and/or
different substrates

Two-Electon Mechanism (Carbanion)


D-Amino acid oxidase (DAAO) catalyzes the oxidation
of D-amino acids to -keto acids and ammonia

Evidence for Mechanism


Ionization of substituted benzoic acids
Hammett Study
Derivation of the Hammett Equation

CO2H+H2O

Ka
X

CO2+H3O+

Scheme 3.36
As X becomes electron withdrawing,
equilibrium constant (Ka) should increase

A Similar Relationship Should Exist for a


Rate Constant (k) where Charge
Develops in the Transition State
Reaction of hydroxide ion with ethylsubstituted benzoates

Et+HO

CO2

CO2+EtOH

Scheme 3.37
As X becomes electron withdrawing, rate
constant (k) should increase

If Ka is measured from Scheme 3.36 and k from


Scheme 3.37 for a series of substituents X, and
the data expressed in a double logarithm plot, a
straight line can be drawn

Linear Free Energy Relationship


Example of a Hammett plot

Figure 3.9

5.0
pNO2
mNO2

4.0

mCl
pCl
mF

3.0

2.0

1.0

pF

oNO2

oF
oCl

H
mCH3
pCH3
pOCH3

oCH3

pNH2

1.0

2.0

log105Ka

3.0

Ortho-substituent points are badly scattered


because of steric interactions and polar effects

Hammett Relationship (Equation)


log k/k0 = log K/K0

(3.3)

log k/k0 =

(3.4)

reaction
constant

- slope
+ slope

electronic parameter
(substituent constant)
carbocation mechanism
EWG +
carbanion mechanism
EDG -

depends on type of
reaction and reaction
conditions

depends on electronic
properties of X

H = 0

Application of Hammett Equation to Study


of an Enzyme Mechanism
D-Amino acid oxidase
H
C
X

H
COOH

NH3+
3.55

= +5.44
X = EWG, Vmax
carbanionic TS

CH2 C
X

COOH

NH3+
3.56

= +0.73
Effect of X diminished
by -CH2-

Proposed Intermediate in the D-amino


Acid Oxidase-catalyzed Oxidation of
Substituted Phenylglycines

COOH

NH3+

COOH

NH3+

3.55

Scheme 3.38
What is the function of the flavin?

C
NH

COOH

Further Evidence for a Carbanion Intermediate


DAAO-catalyzed oxidation of -chloroalanine
under oxygen and under nitrogen
:B

Enz

Fl

Scheme 3.39

H
H2C

COO

H2C

Cl NH3+

Cl NH3

100%N2
irreversible Cl
C

COO

COO

3.60

exclusive
(in N2)

H2C

C
+

COO

O2
+EnzFlH2

NH2
H2O

expected elimination
product
C

H2O2

100%O2
reversible

Cl

NH3+

H3C

EnzFl

3.58

3.57

EnzFl+ H2C

COO

H2O H3C

C
+

COO

NH2

40 : 60
(in air)

H2 C

Cl

COO

3.59

exclusive
(in O2)

Total amount of product(s) is the same under all conditions

Where on the flavin does the nucleophilic


attack occur?
Evidence against C4a addition
Nonenzymatic reaction of benzylamine with N5-ethylflavin
CH3
N
N

O
NCH3

N
Et

CH3

Scheme 3.40

PhCH2NH2
CH3CN

NCH3

N
Et

NH

CH2Ph

No adduct detected enzymatically

Evidence for N5 Addition


Reverse reaction catalyzed by AMP-sulfate reductase
R
N

R
N

+H+

NH

N
O

:SO3=

H
N

NH

N
SO3=

3.61

detected in absence
of AMP

Scheme 3.41

inthepresence
ofAMP

NH

N
H

+ AMPSO3=

Initial Evidence for N5 Attack and for Twoelectron Chemistry


NADH-dependent reduction of 5-deazaflavin
by various flavoenzymes
R
N

O
NH

3.62

5-deazaflavin

O
NH2

+
N
R

various
flavoenzymes

H
N

O
NH

O
NH2

+
N+
R

Scheme 3.42

Comparison of Reduced 5-Deazaflavin


with Reduced Nicotinamide
R
N

Inappropriate
flavin substitute

O
NH

Reduced
5deazaflavin

Favors 2-electron
reactions because
of resemblance to
NADH

Figure 3.10

H
N

N
NH2
H

NAD(P)H

Support for Covalent Carbanionic


Mechanism with DAAO rather than
Electron Transfer Mechanism

H3C

H
N
3.63

H3C

O
N
O

Inverse 2 deuterium isotope effect; therefore


sp2
sp3 in TS, consistent with conversion
to carbanion and nucleophilic addition

Covalent Carbanion versus Radical Mechanisms for


DAAO (Hammett study suggested carbanionic)
R

N
B:

N
NH

N
H

R C

COOH

NH3

R C

COOH

favored
a

+
b H

c
radical
combination

R
N

O
NH

N
O
COOH

: NH2

O
NH

O
R C

NH3

R C

d
+H+,FlH
electron
transfer

COOH

: NH2
+H+,FlH
R C

COOH

NH2

H2O

R C

NH4+

COOH

Scheme 3.43

No base in crystal structure, but -H in line with flavin


Not clear how proton is removed

Carbanion Mechanism Followed by 2 Oneelectron Transfers


Reaction catalyzed by general acyl-CoA
dehydrogenase
O

Fl
SCoA

R
3.68

Scheme 3.46

FlH

SCoA

R
3.69

Initial Mechanism Proposed for Mechanism-based


Inactivation of General Acyl-CoA Dehydrogenase by
(Methylenecyclopropyl)acetyl-CoA

FlH

B:
H
SCoA

3.70

SCoA

Flox

Scheme 3.47
Mechanism-based inactivator

SCoA
3.71

Evidence for Radical Intermediates


Electron transfer mechanism for inactivation of
general acyl-CoA dehydrogenase by
(methylenecyclopropyl)acetyl-CoA
B:
H

only pro-R
removed

SCoA
*

Fl

Fl

SCoA

SCoA
*

Fl
SCoA
3.71

Fl

SCoA
3.72

Both enantiomers inactivate

veryfastno
stereospecificity
(*iseitherRorS)

consistent with a
radical pathway

Scheme 3.48

Other Evidence for Radical Intermediate


Mechanism proposed for formation of 3.73 during
oxidation of (methylenecyclopropyl)acetyl-CoA by
general acyl-CoA dehydrogenase
FAD
O2

SCoA
3.72

SCoA

SCoA
O
3.73

isolated

SCoA

O O

HO

FAD

H+

SCoA
O

SCoA
O

Scheme 3.49

Carbanion Followed by Single Electron Mechanism


for General Acyl-CoA Dehydrogenase
H

:B

O
SCoA

SCoA

H H
B:

NH
O

O
SCoA

SCoA

OH

B H

NH
O

B:

SCoA
H

H
b
R
N
N

B:

O
NH

O
SCoA

R
H

O
NH

N
O

SCoA

O O

NH

a
B:

Not in text

Single Electron Transfer Mechanism


Possible mechanisms for monoamine oxidasecatalyzed oxidation of amines
FlH

Fl

+
RCH NH2
3.75

RCH2NH2
3.74
FlH

Fl

FlH
H

Fl
+
RCH2NH2
3.76

Fl
X

H+

Scheme 3.50

RCHNH2
3.77

X
R

3.78

either Fl- or amino


acid residue

NH2

Mechanism Proposed for Generation of an Activesite Amino Acid Radical during Monoamine
Oxidase-catalyzed Oxidation of Amines
R
N

R
N

NH

N
S

N
H
S

O
NH

Scheme 3.51
Crystal structure of MAO shows no Cys residues
close to the flavin, so this is unlikely
Binda, C.; Newton-Vinson, P.; Hubalek, F.; Edmondson, D. E.;
Mattevi, A. Nature (Struct. Biol.) 2002, 9, 22-26.

Cyclopropylaminyl Radical
Rearrangement

R N

Scheme 3.52

Evidence for Aminyl Radical (radical cation?)


Mechanisms proposed for inactivation of MAO by
1-phenylcyclopropylamine
Fl

Fl
Fl
14Ph

Fl

NH2

14Ph

3.79

3.80

All products derived


from cyclopropyl
ring opening
14Ph

+
NH2

14Ph

3.81

FlH S

+
NH2

Ph

+
NH2

Fl
S

b
Fl
+
B

S
t1/2~80min
pH7.2
H2O

3.85

14Ph

+
NH2

Ph

NH2

+
NH
14Ph
2
3.82
H2O

3.84
H2O

Fl

S
H2O
14Ph

3.87

OH
14Ph

1.NaBH4
2.Raney
Ni

14Ph

O
14Ph

3.86

3.83

Scheme 3.53

Chemical Reactions to Characterize the Structure


of the Flavin Adduct Formed on Inactivation of
MAO by 1-Phenylcyclopropylamine
Fl
NaB3H4

0.5NKOH
O

14Ph

3.85

14Ph

O
3.83
1.CF3CO3H
2.KOH

14PhOH

Scheme 3.54

ca.1equiv3Hincorporation

Baeyer-Villiger reaction

Inactivation of MAO and Peptide Mapping


CH3

Cys-365
N
H

Ph

LysLeuXAspLeuTyrAlaLys
3.89

3.88

MALDI-TOF gives mass


corresponding to X as
HO

S
Cys

Mechanism Proposed for Inactivation of MAO by


N-cyclopropyl--methylbenzylamine
CH3
Ph

Fl

CH3

Fl

N
H

Ph

CH3

N
H

Ph

3.88

HO

N
H

CH3

Fl

Fl
H+

Ph

N
H
S

NaBH4 O

CH3

H2O

Ph
CH3
Ph

Scheme 3.55 (modified)

NH2

N
H

+H+
S

Further Evidence for Aminyl Radical


(radical cation?) Intermediate
Mechanism proposed for MAO-catalyzed
oxidation of 1-phenylcyclobutylamine and
inactivation of the enzyme
Fl

Fl
b

Ph

NH2

Ph

3.90

Ph

NH2

Fl

Fl
a

+
NH2
3.91

Ph

3.94

b
FlH

Ph

Fl

O
Ph

N
3.93

+
NH2

Ph

N
H
3.92

N t Bu
EPRspectrum
(tripletofdoublets)

Scheme 3.56

Evidence for -Carbon Radical Intermediate


Oxidation of (aminomethyl)cubane by MAO
Gives product of a cubylcarbinyl radical intermediate
NH2

Fl

Fl

NH2

H+
a

NH2
3.96

3.95
Fl
FlH
CHO

FlH

Fl

c
NH2

NH2

3.98

3.97

detected

Scheme 3.57

furtherdecomposition
andinactivation

Reactions to Differentiate a Radical from


a Carbanion Intermediate
O
O

O
B

Scheme 3.58

R
R

Further Evidence for -Carbon Radical with MAO


Mechanism proposed for MAO-catalyzed
oxidation of cinnamylamine-2,3-epoxide
Ph

Fl

O
3.99

Fl
Ph

NH2

FlH
H2O

Ph

NH2

isolated
HOCH2CHO
+
PhCHO

H+

Ph

NH2

NH2

Fl
Ph

Scheme 3.59

No products of a two-electron
epoxide ring opening detected

NH2
O

More Evidence for -Carbon Radical

Mechanism proposed for MAO-catalyzed


decarboxylation of cis- and trans-5-(aminomethyl)-3(4-methoxyphenyl)-2-[14C]dihydrofuran-2(3H)-one
NH2
Ar

14

NH3

Fl

Fl

Ar

14

NH2

H+
Ar

O
3.100

3.101
Fl

O
H
3.102

isolated

Ar

14

Fl

+H+,+H2O
NH3

evidence for reversible e- transfer

(Fl Fl- , Fl- Fl)

NH2
Ar

14CO2

detected
3.101a

Scheme 3.60

Evidence for a Covalent Intermediate


Mechanism proposed for inactivation of MAO by (R)- or
(S)-3-[3H]aryl-5-(methylaminomethyl)-2-oxazolidinone
NHMe
ArCxH2O

ArCxH2O

N y O
3.103

NHMe

Fl

Fl

N y O
O

H+
X

NHMe

NHMe
X

ArCxH2O

N y O
3.104

ArCxH2O

O
+
NHMe

N y O

ArCxH

2O

N y O

Fl
FlH

Scheme 3.61
When x = 3 and y = 14, both radiolabels
are incorporated into the protein
O

Example of a Hydride Mechanism


Reaction catalyzed by UDP-Nacetylenolpyruvylglucosamine reductase (MurB)
2nd step in bacterial peptidoglycan biosynthesis
OH
HO
OOC

MurB

O
O

NH

NADPH
H+
UDP

3.105

EP-UDP-GlcNAc

Scheme 3.63

OH
HO
NADP+

O
OOC

NH

UDP

3.106

UDP-N-acetylmuramic acid

Hydride Mechanism for a Flavoenzyme


(MurB)
H
R
N

EPUDPGlcNAc

NH

N
H

R
N

NADP+

OH
M+

HO

O
B:
H

NH

UDP

OH
HO

M+ O

Scheme 3.64

FAD

NH

NH2

In situ generation
of FADH

O 229Ser

O
B+

NH

3.105

UDP

OH
HO

M+ O

O
O

O
3.106

NH

UDP

Evidence for the Hydride Mechanism


MurB-catalyzed reduction of (E)-enolbutyryl-UDPGlcNAc with NADP2H in 2H2O
HO
OOC

CH3

OH

OH
O
O
O

3.107

extra Me for
stereochemical
determination

NH

MurB
O

UDP

NADPD
D2O

HO

O
O
O
D

NH

UDP

H
CH3

3.108

anti-addition

Scheme 3.65
A radical mechanism is not
expected to be stereospecific

Determination of the Stereochemistry of 3.108


Conversion to 2-hydroxybutyrate of the product
formed from MurB-catalyzed reduction of (E)enolbutyryl-UDP-GlcNAc with NADP2H in 2H2O

D-configuration
OH

3.108

NaOD

HO

O
O

D
D

H
CH3

Scheme 3.66

NH

OH
alkaline
phosphatase
O PO3=

HO

O
NaOD

O
NH

D O
D

H
CH3

OH

OH
D

D
CH3
3.109

Substrate for D-lactate


dehydrogenase but not
L-lactate dehydrogenase,
therefore 2R stereochemistry

Enzymatic Syntheses of (2R,3R)- and (2R,3S)isomers of 2,3-[2H2]hydrobutyrate for NMR


Comparison with 3.109
pyruvate
kinase

O
O
O

H3 C

D2O

omit ATP

O
H

O
O

pyruvate
kinase
H2O

NADD

Scheme 3.67

Dlactate
dehydrogenase

OH
O

(2R,3R)2,3[2H2]2
hydroxybutyrate
O

H3 C
H

H3C

D2O

pD7

H3C

D
O

NADD

H3C

Dlactate
H
dehydrogenase

OH
O

(2R,3S)2,3[2H2]2
hydroxybutyrate

Stereochemistry of the MurB-catalyzed


Reduction of (E)-enolbutyryl-UDP-GlcNAc
R
N

O
HN

M+
B+ H O
RO
O

HN

Ser229

reface

B:

M+
O

RO
O

Ser229

O
RO

Scheme 3.68

H
O

R
N

H
R

Reaction Catalyzed by Dihydroorotate


Dehydrogenase
:B
O
HN
O

N
H
3.110

H
H
COOH
H

O
HN
O

+
N
H

FlH

COOH

Fl

Scheme 3.69

D isotope effects on both Hs; therefore concerted

Unusual Reaction Catalyzed by a Flavoenzyme


UDP-galactopyranose mutase (UGM)

Requires FAD; only reduced enzyme is active


When UGM was incubated with UDP-[3H]-galactopyranose and treated with
NaCNBH3, enzyme was inactivated (not when NaCNBH3 was omitted);
gel filtration gave radioactive enzyme
Acid denaturation precipitated protein and all tritium released; flavin
fraction in supernatant was tritiated
Mass spectrum consistent with a flavin-galactose adduct
Absorption spectrum characteristic of N5-monoalkylated flavin
pKa of N5 of reduced FAD is 6.7, suggesting can be deprotonated
Soltero-Higgin, M.; Carlson, E. E.; Gruber, T. D.; Kiessling, L. I. Nature Struct. Mol. Biol. 2004, 11, 539-543

UDP-galactopyranose mutase (UGM)


UGM reconstituted with 5-deazaFAD is inactive.1
2- and 3-F UDP-galactopyranose are substrates; excludes a mechanism
involving oxidation at C2 or C3.2
Rate of 2-F UDP-galactopyranose as substrate is 1/750 that of substrate;
rate of 3-F UDP-galactopyranose as substrate is 1/4 that of substrate.
Supports a mechanism with an oxocarbenium ion at C1 (SN1 mechanism)

Huang, Z.; Zhang, Q.; Liu, H.-w. Bioorg. Chem. 2003, 31, 494-502.

Zhang, Q.; Liu, H.-w. J. Am. Chem. Soc. 2001, 123, 6756-6766.

Mechanism of UDP-galactopyranose mutase (UGM)

Mansoorabadi, S. O.; Thibodeaux C. J.; Liu, H.-w. J. Org. Chem.. 2007, 72, 6329-6342.

Artificial Enzyme (Synzyme)


Synthesis of flavopapain
Me
N
S

Br

Me
N

papain

Scheme 3.70

NH

N
O

S
O

NH

N
O

3.111

catalyzes oxidation
of NADH to NAD+

Unusual Reaction Catalyzed by Urate Oxidase


No flavin, but substrate reacts like a flavin
H
N

H
N

O
N
H

compare
structures
R
N
N
H

O
NH

O
3.112

H2O2
H2O

H
N

O
N
H

O
NH

reduced flavin

H
N

O
NH

HO O
3.113

detected

H
N

O2

comes from H2O,


not O2 (using 18O)

H
N

O
N
H

O
NH2

3.114

Scheme 3.71

Mechanism for an Oxidase-catalyzed Oxidation


of Reduced Flavin to Oxidized Flavin for
Comparison with Urate Oxidase
R

N
N
H
B H

NH
a

O
B

3.54

Flox

H O O
OH
3.53
H2O2

radical
combination
N

O
O O

NH

N
H

H2O2

O O

e transfer

R
N

O
NH

O O

2ndetransfer
+H+
H

Scheme 3.33

Possible Mechanism for the Urate


Oxidase-catalyzed Oxidation of Urate
H
N

H
N

O
N
H

probablyby
two1esteps

H
N

NH

NH

3.112

B:

H2O2

H
N
O

O
NH

O O
OH

detected

O
OH

B:

Scheme 3.72
Just like mechanism for oxidation
of reduced flavin by O2

3.113

Pyrroloquinoline Quinone Coenzymes (PQQ)


HOOC

1
HN

2 COOH

8
HOOC

4
N
6

O
3.115

Bound to quinoproteins
Also called methoxatin, coenzyme PQQ

Possible Mechanisms for the Glucose


Dehydrogenase-catalyzed Oxidation of Glucose
OOC

Nucleophilic
mechanism

HN

A
OOC

N
Ca2+

from model study


with MeOH C-5
favored over C-4
addition

OH

O
Ca2+ H
O
OH

O
OH

HO

HO

144His

..

COO

HN

N
Ca2+

H
O

144His

..

OH

HO

OOC

OOC

OH

COO

HN

B
OOC

OOC

OOC

OOC

O
144His

..

HN

OH

144His

..

HO

144His

..

OOC

OOC

COO

HN

OH

Ca2+

144His

..

O
OH

HO

Ca2+

COO

O
O

OH

HO

N
Ca2+

OH

HO

OOC

COO

HN

OH

HO

OOC

Hydride
mechanism

OH
HO

COO

Scheme 3.73

HO

From crystal structure, hydrogen over C-5 carbonyl, suggesting hydride mechanism

Evidence for Nucleophilic Mechanism for Plasma Amine Oxidase


Plasma amine oxidase

(contains CuII)

Schiff base mechanism proposed -- NaCNBH3 inactivates the enzyme in the


presence of substrate

14Ph

originally thought it
was a PQQ enzyme
(We will see it is not)

NH2

+NH

H isotope
effect

+NH

14Ph

H
:B

NaCNB3H3

OH

B+
14Ph

H2O

3H+
OH
NH
14Ph

1 equiv. 14C
no 3H from
NaCNB3H3

OH
3H

O
NH

NH3+
14PhCHO

NH2

+
HN

14Ph

Therefore excludes oxidation to 14PhCHO


followed by Schiff base formation with a Lys

14Ph

NaCNB3H3

+
H2 N

3H

14Ph

Scheme 3.74

Isotope Labeling Shows Syn Hydrogens are


Removed (one-base mechanism)
Stereochemistry of the reaction catalyzed by plasma amine oxidase (PAO)

PQQ is not the actual cofactor for PAO

COOH
NH

COOH

N
HN +
1
2

HS

HS
HR
HR
Ar

COOH
:B

Scheme 3.75

HN +

HR HS

+B

HS

O
HN +

HR

HR
HS

Ar

HR

HS

Ar

HS

HN

HR
Ar

:B
HS

Topa Quinone (TPQ), 6-Hydroxydopa,


is the Actual Cofactor for PAO
O
Leu

Asn

NH

CH

Asp

Tyr

CH2
O

2
3

OH
3.116

Characterized by Edman degradation, and mass,


UV-vis, resonance Raman, and NMR spectrometries

Using
NH a Hammett study showed
X
= 1.47 0.27 (carbanion-like TS)
2

Plasma amine oxidase-catalyzed amine oxidation


with topa quinone shown as the cofactor
CH2
O

NH2

O
O

+
N
H

3.117 H

H
H
R

OH

N
H

RCHO
NH2

Scheme 3.76

:B

OH
3.118

H2 O
OH

+
N
H

Model Study for Topa Quinone


O
HN

R
NH

R= tBu 3.120
iPr 3.121
3.122
Et
Me 3.123
O

O
O

R= OMe
Me
H
O

3.124
3.125
3.126

OH
O

OMe

OH
3.119

C-5
3.127

R= H
OMe

3.128
3.129

O
O

Preferential attack at C-5 carbonyl by nucleophiles


Resonance Raman spectrum shows carbonyl at C-5 has greater
double bond character (more reactive) than at C-2 or C-4

Chemical Model Study for the Mechanism


of Topa Quinone-dependent Enzymes

NH2

O
OH

O
O
H3N+

Scheme 3.77

O
O
H3N+

Deactivates C-2
and C-4 carbonyls,
so C-5 carbonyl is
more reactive

Detailed Mechanism Proposed for


Topa Quinone-dependent Enzymes
Mechanism for Plasma Amine Oxidase
CH2

CuII
OH2

CuII
Ph

NH2

H2O

CH2

:B

H O

CuII

H
+
N

CH2
H

CHPh

NH

OH

CHPh

H2O
PhCHO
NH3

H2O
CuII

CH2

H2O2

OH2 O

Scheme 3.79

O2

H O

+
NH2

CH2

CuII
H

O
NH2
OH
3.131

Mechanism Proposed for Reoxidation of


Reduced Topa Quinone
Based on EPR spectroscopy

CuII
H

CH2

CuI

CH2

2H+

O
NH2
OH
3.131

Scheme 3.80

OH

O2

H2O2

CH2

CuII
OH2

NH
O

H
3.132

detected

+
NH2

Mechanism Proposed for Biosynthesis of Topa


Quinone from Tyrosine
H+

CuII
CuII

CuI

OH

CuI

OH

O
O2

CuII
O

CuII
O

CuII
O
O

H O

CuII
O
O

B:

CuII

H+

CuII

O2,H+

O
O

O
CuII

H
B:

H2O2

OH
O

Topa quinone is ubiquitous - found


in bacteria, yeast, plants, mammals

O
TPQ

Scheme 3.81

Tryptophan Tryptophylquinone Coenzyme

Protein

NH

Protein

Observed by
X-ray analysis
O

N
H
O
3.133

in methylamine dehydrogenase
Hammett study with
X

NH2

+ (carbanion
mechanism)

Coenzyme in Lysyl Oxidase

AspThr(modifiedTyr)AsnAlaAsp
ValAlaGluGlyHis(modifiedLys)
3.134

Isolated from a proteolytic digestion

Structure of Lysine Tyrosylquinone


in Lysyl Oxidase
ValAlaGluGlyHis

AspThr N CHCONH
H
NH
CH2
H C
CH2CH2CH2CH2 NH
O

OH
O

Lys

3.135

Tyr

AsnAlaAsp

Enzymes Containing Amino Acid Radicals


Mechanism proposed for galactose oxidase using a covalently bonded
cysteine cross-linked tyrosine radical
Tyr272
O.
Cys228

OH

Tyr
O . Cu(II)++

Cu(II)++

Cys

3.136

H+

HOO

O.

O2
Cu(II)++

Tyr

O
H

O . Cu(II)++

Cu(II)+
O

Cys

Hatomtransfer
stepwisemechanism
Tyr

Tyr

Cu(I)+
Cys

ER2;radicalE2
concertedmechanism

O2

H
H

O2
OH

Scheme 3.82

Cys

O
H

Cys

Tyr

Tyr

O H
Cys

RCHO

Tyr

OH

OH Cu(I)+
O
H

OH
Cys

Cu(II)++

H . R
ketyl
radicalanion

Mechanism-based Inactivation of Galactose


Oxidase by Hydroxymethylquadricyclane and
Hydroxymethylnorbornadiene
Tyr272

CH2OH

CH2O

Cys228

Tyr

Scheme 3.83

O . Cu(II)++

3.136

Cys

OH Cu(II)++

3.137

quadricyclane
analogue
CH2OH

O
C

O
C

ketyl radicals
O

sameaswith3.137

3.138

norbornadiene
analogue

Tyr

kH/kD = 6 on inactivation

3.139

Cys

[,-2H2] 3.137

OH Cu(II)++

inactivatedenzyme
complex

1e- reduced form

Iron-sulfur Clusters and Pyridoxamine 5-Phosphate (PMP)


Biosynthesis of ascarylose
Reaction catalyzed by CDP-6-deoxy-L-threo-D-glycero4-hexulose-3-dehydratase (also called E1) and CDP-6deoxy-3,4-glucoseen reductase (also called E3)
NH2
HO

Me

O
OH
HO

NAD+

OCDP
OH
3.140

HO

O
OH

Me
OCDP
OH
3.147

ascarylose

NADPH

OH

=O PO
3

Pyr=pyridineringofPMP

Me

Me
OCDP
OH
3.146

3.142

O
OCDP
OH
3.145

H
N
+

H2O

OCDP
OH

Pyr

O
O

O
OH

H
N
+

3.142

E1

Me

Me

OCDP
OH
3.141

(PMP)

Pyr

3.143

E1/E3

OCDP
OH

NADH,FAD
Fe(III)Fe(II)S2
Me

H
N
+
Pyr

O
OCDP
OH

3.144

Scheme 3.84

Pyridoxamine 5-Phosphate (PMP)


CH2NH2
OH

=O PO
3

CH3

3.142

Usually in carbanionic reactions of amino acids


With E1/E3 PMP may be involved in two oneelectron reductions (EPR)

Iron-sulfur Clusters
Cys S

Cys S
Fe
Cys

S Cys
Fe

[2Fe-2S]

Fe
S

3.148

Cys S

Cys

Cys S

S
Fe

Fe
S

Fe
S

Cys

Fe
Fe

Fe

S Cys

Cys S

3.149

3.150

[3Fe-4S]

[4Fe-4S]

1 electron and 2 electron transfers

Cys

Mechanism Proposed for the Reduction of


CDP-6-deoxy-3,4-glucoseen by E1 and E3
=O

E1

Me
O

OH

PMP

OCDP
OH

3PO

H
HN +
Me

=O

B:
Me

O H

=O

OH

Me

OCDP
OH

O H

N
+

Me

HN +

OCDP
H OH
B

Me

+
N
O H

OCDP
OH

FADH

NAD+
E1,PMP
E3,NADH

3PO

Me

HN +

O
N
+

3PO

**

NADH
FAD

Fe(III)2S2
E3

E3

Fe(III)Fe(II)S2
FADH

Fe(III)Fe(II)S2
E1

H+

Fe(III)2S2

1e- transfer
Fe(III)Fe(II)S2
=O PO
3

Me
O
O
OCDP
OH
3.145

H2O
PMP

Me

E3
E1

Me
HN +

Fe(III)2S2

+
N
H

O
OCDP
OH

* In 3H2O, 1 3H in product
** (4R)- and (4S)-[4-3H]NADH both transfer 3H
3
H released as 3H2O

Fe(III)Fe(II)S2
Fe(III)2S2

1e- transfer

=O PO
3

Me

HN +
Me

+
N
O H
3.151

O
OCDP
OH

EPR evidence

Scheme 3.85

Molybdoenzymes and Tungstoenzymes


OH OH
O
O
HN
H2N

H
N

O
MoVI

H2N

N
H

HO

OPO3=

HN
N

H
N

N
H

HN

3.152

N
H

O3

S
O
HN
H2N

H
N
N
H

O
O

MoVI

H
N

N
H

WVI

S
O

P
O

3.153

=PO

NH2
NH

O
N

H2N

H
N

O
O

P
O

N
O

OH

NH
N

NH2

OH

NH2
NH

S
OPO3=

O
3.154

Hydroxylation generally by flavin, heme, pterin enzymes (next chapter)


with the O coming from O2; in these enzymes, the O comes from H2O

Mechanism for Sulfite Oxidase (in liver)


O
O

:S
O
O
HN
H2N

H
N

O
MoVI

O
HN

O
OPO3

N
H HO

H2N

H
N

O
MoVI
S

H
N

N
H HO

HN
H2 N

O
2e

HN
H2 N

Scheme 3.86

H
N

N
H HO

O
H O

IV
S Mo
S

SO4=
OPO3=

H2N

O
S

IV
S Mo
O
S

H
N

N
H HO

HN

OPO3=

O from H2O
O

IV
S Mo
O
S

3.152

OPO3=

N
H HO

O
H OH

B:

OPO3=

Reduction with No Cofactors


Hydrogenases
The only known non metallohydrogenase
Reduction of N5,N10-methenyl tetrahydromethanopterin to
N5,N10-methylene tetrahydromethanopterin catalyzed by the
hydrogenase from a methanogenic archaebacterium
H2N

H
N

CH3
H

HN

N
O
H

H2N

+
14a

CH3

+H2

Scheme 3.89

H
N

CH3
H

HN

N
O

3.158

CH3

H+

N
HR H
S

pro-R
specific

3.159

Model Study for Metal-free Hydrogenase


Reaction of perhydro-3a,6a,9a-triazaphenalene
with tetrafluoroboric acid
N
N

N
H
3.161

+H+

strong acid

antiperiplanar stereoelectronic
effect

Scheme 3.91

110 C

3.162

irreversible

+H2

Mechanism Proposed for Oxidation of


N5,N10-methylene tetrahydromethanopterin to
N5,N10-methenyl tetrahydromethanopterin
(reverse of the reaction in Scheme 3.89)

H
HR
H3C H R
N
H

ring

HS
ring

3.159

Scheme 3.90

H H

H3C H R
N
H

initially, not resonance stabilized


+
N

ring
3.160

ring

H3C
H

+
N

ring

conformational
change

3.158

H +H2
ring