Transcript The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation Redox Without a Coenzyme Internal redox reaction.

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
O
Ph C H +
Ph C H
-OH
Ph COOoxidized
HOO
OPh 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
HO
O
+ GSH
CH3 C
C
3.3
H
glyoxalase I
SG
3.4
HO
CH3 C
OH
O
C
SG
H 3.4
Scheme 3.3
+
H2O
glyoxalase II
CH3 CHCOO-
+ GSH
Glutathione (GSH)
COO-
O
CH2SH
H3N CHCH2CH2 CNH CH C NHCH2CO2(-Glu-Cys-Gly)
3.3
O
Hydride Mechanism for Glyoxalase
H B
BO O
CH3
C C H
H SG
CH3
oxidized
reduced
O
O-
C
C SG
OH O
CH3
C
C SG
H
H
H2O
OH
GSH +
CH3 CH
glyoxalase II
COO-
Scheme 3.4
Intramolecular Cannizzaro reaction
• 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
O
O
C
C
H
+ GSH
CH3
B:
H
O
OH
C
C
H
SG
CH3
HO
O
C
C
B+ H
SG
3.5
B:
Scheme 3.5
cis-enediol
H
no exchange
with solvent
CH3
HO
O
C
C
H
SG
Reaction of Glyoxalase with
Fluoromethylglyoxal
Another test for the mechanism
O O
FCH2C CH
HO
O
C
C
O
O
CH3C
C
glyoxylase
3.6
Scheme 3.6
GSH
FCH2
H
SG
+
3.8
3.7
same oxidation state
SG
Hydride Mechanism for the Reaction of
Glyoxalase with Fluoromethylglyoxal
O O
FCH2C CH
B+ H
O
O-
FCH2C
C
HO O
F
GSH
3.6
SG
H
H
CH2
C
O
CSG
O
O
CH3C
C
3.8
B:
3.7
Scheme 3.7
HO
CH2 C C SG
SG
Enediol Mechanism for the Reaction of
Glyoxalase with Fluoromethylglyoxal
B+ H
O
O-
FCH2C
C
O O
FCH2C CH
GSH
3.6
HO
SG
F CH2 C
OF
C SG
HO
O
C
C
CH2
a
b
H
B+ H
B:
b
B+
SG
H
a
O
O
CH3C
C
3.8
Scheme 3.8
SG
CH2
HO
O
C
C
SG
FCH2
HO
O
C
C
H
3.7
SG
Hydride Mechanism for the Reaction of Glyoxalase
with Deuterated Fluoromethylglyoxal
B+
O
O O
FCH2C CD
H
GSH
3.9
FCH2C
HO
OC
SG
F
CH2
C
O
C
SG
O
O
CH3C
C
-F-
D
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
O-
FCH2C
C
O O
FCH2C CD
GSH
3.9
O-
HO
SG
F CH2 C
C SG
F
HO
O
C
C
CH2
B+ D
B:
B+
b
-F-
O
O
CH3C
C
a
b
D
SG
CH2
HO
O
C
C
SG
SG
D
a
FCH2
HO
O
C
C
SG
D
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 O
O O
FCH2C CH
FCH2C CD
yeast
32.2 ± 0.2
40.7 ± 0.2
rat
7.7 ± 0.1
13.3 ± 0.9
mouse
26.4 ± 1.0
34.8 ± 0.5
yeast/D2O
33.8 ± 0.2
39.1 ± 0.4
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)+
NAD(P)H
NH2
N
N
N
NH2
N
CH2
OR' HO
O
O O
OP OP O CH2
O-
OHO
H
N
O
O
NH2
N
N
N
N
CH2
OR' HO
O
O O
OP OP O CH2
O-
a, R' = H
b, R' = PO3=
O-
N
O
HO
OH
3.10
H
NH2
O
OH
3.11
Enzyme without coenzyme bound - apoenzyme
Enzyme with coenzyme bound - holoenzyme
apoenzyme
coenzyme
holoenzyme
Called
reconstitution
Abbreviated Forms
O
H O
H
NH2
NH2
N
N
R
R
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
NH2
=
O3PO
COOH
PPi
O
=
O3PO
N
O
ATP
N
3.14
OP2O6-3
OH OH
3.15
N
PPi N
+
N
OH
O
OH
N
OH
CH2
OH HO
O
3.16
nicotinic acid
(vitamin B3)
niacin
O O
OP OP O CH2
O
O- OHO
from ATP
3.17
N
OH
Gln
ATP
NH2
O
N
N
N
N
NH2
O O
CH2 OP OP O CH2
O
O- OOH HO
O
HO
N
OH
3.18
Scheme 3.11
Reactions Catalyzed by Pyridine
Nucleotide-containing Enzymes
H
C
C
OH
O
Oxidation
potential
NAD+/NADH
is -0.32 V
H
C
C
+NH3
O
C
H
C
O
Figure 3.1
O
O
C
C
H
H
C
N
H
H
C
C
C
N
Reactions Catalyzed by Alcohol
Dehydrogenases
Mechanism
B:
H
R C
R C
O H
H
+B
H
H
O
O
H O
H
NH2
NH2
N
:
R
R
N
In 3H2O, no 3H in NAD(P)H
Hydride mechanism
Scheme 3.12
Reaction Catalyzed by Alcohol
Dehydrogenases Using Labeled Alcohol
H
O
NH2
RC *H2OH +
N
R
Scheme 3.13
H2O
H
O
R
C
*H
*H O
NH2
+
N
R
No *H found in H2O
Supports hydride mechanism
Test for a radical intermediate
Cyclopropylcarbinyl Radical Rearrangement
k = 108 s-1
3.19
Scheme 3.14
3.20
Test for the Formation of a Radical
Intermediate with Lactate Dehydrogenase
O
OH
CO2H
3.21
pig heart
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
OSnBu3
CO2Me
Bu3SnH
AIBN

OSnBu3
CO2Me
CO2Me
Bu3SnH
Scheme 3.16
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
O
NADH
Ph
CH2Cl
3.23
Scheme 3.18
Ph
CH3
3.24
radical reduction
product
Horse Liver Alcohol Dehydrogenase-Catalyzed
Reduction of -Haloacetophenones
O
OH
HLADH
Ph
CH2X
Scheme 3.19
X
NADH
Ph
*
3.25
hydride reduction product
(stereospecific)
X = F, Cl, Br
Supports no radical intermediate
O
When X = I, get mixture of 3.25 (X = I) +
Electron transfer is possible
if the reduction potential is
low enough
Ph
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
H
C
COO-
A
Figure 3.2
D
B
lowest priority behind
counterclockwise
(S)
Determination of Prochirality
Caacd
prochiral
Cabcd
chiral
pro-R hydrogen
H
2H
H
CH3
OH
H
CH3
OH
chiral
prochiral
R
pro-S hydrogen
H
CH3
Figure 3.3
H
H
OH
prochiral
2H
CH3
OH
chiral
S
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
si face
si face
CH2
O
CH3
C
re face
Figure 3.4
H
CH3
C
re face
H
Reaction of Yeast Alcohol Dehydrogenase
(YADH) with (A) [1,1-2H2]ethanol and NAD+
and (B) Ethanol and [4-2H]NAD+
H
O
D
NH2
A
+
CH3CD2OH
H
O
YADH
NH2
N
N
R
R
+
CH3CDO
3.26
D
O
H
NH2
B
N
R
+
CH3CH2OH
D
YADH
NH2
N
R
3.27
Scheme 3.20
O
+
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
H
No 2H
O
H
NH2
A
YADH
+ CH3CHO
stereospecific
O
H
NH2
+
N
H
D
3.28
R
No H
O
D
NH2
B
D
N
R
3.26
O
YADH
+ CH3CHO
N
NH2
+
+
3.26
N
R
R
3.27
H
C
3.28
CH3COH
NH2
+
Scheme 3.21
O
N
R
YADH
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
D
YADH
A
O
pro-R
NH2
CH3CD2OH + NAD+
+ CH3CDO
N
R
3.26
D
O
B
3.26 + H2C
G3PDH
NH2
CH C OP
OP OH
O
+
+N
R
pro-S transferred
Scheme 3.22
H2C
CH CHO
OP OH
3.30
+ Pi
Transition State for Hydride Transfer
Anti- and syn- conformations of NADH
Figure 3.5
HR
HS O
O
HR
NH2
N
RO
H2N
:
N
RO
anti conformation
syn-axial
electrons
assist
:
O
O
H
OH OH
HS
H
OH OH
syn conformation
pro-S
transfer
pro-R
transfer
Boat-like TS‡
The enzyme may drive equilibrium
Boat-boat equilibria of NADH
anti-NADH
HR
HS
HR
CONH2
CONH2
HS
N
RO
N
RO
O
HO
O
OH
OH
HO
HR transfer
HR
syn-NADH
HS
HS
H2NOC
HR
H2NOC
RO
N
O
Figure 3.6
HO
N
RO
O
OH
HO
OH
HS transfer
Oxidation of Amino Acids to Keto Acids
Possible mechanism for the reaction catalyzed
by glutamate dehydrogenase
K125
NH
.. 2
Hydride transfer
H
CONH2
+N
R
-OOC
H
+
NH2
K125
D165
H
H
H
OH
+
H OOC D165
NH2
CONH2
CO2H3N K113
CO2-
NH
.. 2
CO2-
N
R
COO-
H3N K89
H3N K113
H3N K89
K125
K125
HOOC
NH3
O
NADPH
D165
NH3
CO2-
H
O
-
+ OOC
NH3
D165
CO2H3N K113
CO2-
Scheme 3.24
NH3
H3N K89
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
H
B:
O
H
–S
O
O
S
A
R
R
H
R
covalent catalysis
H
3.31
H
3.32
NAD+
O
H
B–
+
R
H
3.33
Scheme 3.25
O H
O
OH
RCHO + H2O
S
Hydride transfers
B:
B
+ NADH
O
R
NAD+
via hydrate
OH
NADH
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:
O
N
H
N
HN
H
B
O
O
N
N
N
X
RP
RP
H
H O
NH2
inosine MP
NH2
N+
N
R
R
O
N
H
O
X H
B:
O
B:
N
HN
Scheme 3.27
H B+
H OH HN
N
N
X
RP
:B
N
O
X
xanthine MP
B+
H
:B
N
HN
H
RP
3.37
H
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
N
COOH
D
OH
3.40
Scheme 3.28
D
Urocanase Reaction Run with a [13C]
Pseudo-substrate
apo-urocanase reconstituted
with [13C]NAD+ O
“substrate”
13
13
NH2
H
N
COOH
N+
N
H
3.41
exchangeable
proton
reduced
side chain
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
solvent incorporated
B
H
H
N+
COO-
H
N
:
N
H
COOH
O
O
COO-
:
N
N
:B
N
H
NH2
N
N+
exchangeable
R
R
H
COO-
N
N
H
OH
:
+ NAD+
COO-
+N
COO-
N+
N
OH
N
R
H
H
N
oxidative quench oxidizes
this reduced adduct
O
NH2
NH2
When 3.41 is used,
the reaction stops here.
N
R
H
OH
O
NH2
H
B+
O
H
B:
NH2
N
R
Scheme 3.29
Flavin Coenzymes
Biosynthetic conversion of riboflavin
to FMN and FAD
NH2
N
O
CH2O
CH2OH
(CHOH)3
8a
8
7
9
6
(CHOH)3
O-
CH2O
O-
N 10a N
10
O ATP
NH
ADP
N
riboflavin
(vitamin B2)
Scheme 3.31
N
O ATP
PPi
N
O
O-
N
O
FMN
O
3.50
FAD
N
O CH2
O
O-
O
NH
N
3.49
P
HO
NH
N
O
CH2
N
O
3.48
P
(CHOH)3
CH2
CH2
N 4a
5
P
O
OH
N
Interconversion of the Three Oxidation
States of Flavins
oxidized
semiquinone
reduced
R
R
N
N
O
+1e-1e-
NH
N
O
(Fl)
N
N
O
NH
N
3.51
some covalently attached to
The protein at these positions
R
-1e-
N
N
N
N
N
H
3.52
FlH
O
Fl
O
NH
O
N
H
N
N
H
O
O
Scheme 3.32
O
R
R
N
_
N
+1e-
O
N
H
Redox Reactions Catalyzed by Flavindependent Enzymes
H
C
C
OH
O
H
C
C
NH2
O
CH2
CH2
+
C
CH
O
NADH
HS
SH
Figure 3.8
NH4+
CH
C
O
NAD+
S
S
Oxidases vs. Dehydrogenases
Mechanisms for an oxidase-catalyzed oxidation of
reduced flavin to oxidized flavin
R
R
N
N
O
N
N
O
a
N
H
B H
NH
O
b
O O
B
-H2O2
NH
N
a
Scheme 3.33
Flox
H O O
OH
3.53
only if spin
inversion
occurs
b
-H2O2
O O
e transfer
radical
combination
R
N
N
3.54
d
NH
N
H
O
O
O O
c
2nd e- transfer
+ H+
H
B
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
O
N
NH
B
H
Acceptor
Scheme 3.34
N
O
NH
N
O
Acceptor
R
N
N
NH
N
O
O
Acceptor
O
Mechanisms for Flavoenzymes
Overall reaction of flavoenzymes
Substrate
Enzyme-FlH-
+
Enzyme-Flox
+
Scheme 3.35
Acceptor
(O2)
Enzyme-FlH-
Oxidized substrate
(product)
+
Enzyme-Flox
Reduced acceptor
(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
Ka
CO2- + H3O+
CO2H + H2O
X
X
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
CO2Et +
X
HO-
k
CO2- + EtOH
X
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
p-NO2
m-NO2
4.0
m-Cl
p-Cl
m-F
3.0
p-F
o-Cl
lo
g
10
5
k
2.0
1.0
o-NO2
o-F
H
m-CH3
p-CH3
p-OCH3
o-CH3
p-NH2
1.0
2.0
log
105
3.0
Ka
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
H
C
X
COOH
NH3+
C
X
COOH
NH3+
C
X
3.55
Scheme 3.38
What is the function of the flavin?
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
C
COO-
H2C
C
Cl NH3+
Cl NH3
100% N2
irreversible -ClC
COO-
COO-
O
3.60
exclusive
(in N2)
H2C
C
+
COO-
O2
+ Enz-FlH2
NH2
H2O
expected elimination
product
C
H2O2
100% O2
reversible
Cl
NH3+
H3C
Enz-Fl
3.58
3.57
Enz-Fl + H2C
COO-
H2O H3C
C
+
COO-
NH2
H2C
C
Cl
O
COO-
3.59
40 : 60
(in air)
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
O
Scheme 3.40
N
N
O
PhCH2NH2
CH3CN
NCH3
N
Et
O
NH
CH2Ph
No adduct detected enzymatically
Evidence for N5 Addition
Reverse reaction catalyzed by AMP-sulfate reductase
R
R
N
N
O
+H+
NH
N
SO3=
SO3=
O
NH
N
O
:
N
H
N
O
3.61
detected in absence
of AMP
Scheme 3.41
in the presence
of AMP
R
H
N
N
NH
N
H
O
O
+ AMP-SO3=
Initial Evidence for N5 Attack and for Twoelectron Chemistry
NADH-dependent reduction of 5-deazaflavin
by various flavoenzymes
R
N
N
O
NH
3.62
H
H
O
R
O
N
NH2
+
N
R
H
H
N
various
flavoenzymes
O
NH
H
H
O
NH2
+
N+
R
O
5-deazaflavin
Scheme 3.42
Comparison of Reduced 5-Deazaflavin
with Reduced Nicotinamide
R
H
N
N
Inappropriate
flavin substitute
O
NH
H
H
Reduced
5-deazaflavin
O
R
Favors 2-electron
reactions because
of resemblance to
NADH
N
NAD(P)H
NH2
H
Figure 3.10
H
O
Support for Covalent Carbanionic
Mechanism with DAAO rather than
Electron Transfer Mechanism
H
O
H
N
N
H3C
O
O
H3C
O
3.63
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
O
O
a
b
R C
COOH
favored
a
N
+
b -H
c
R
N
NH
COOH
: NH2
COOH
+H+, -FlH-
O
d
O
NH
: NH2
radical
combination
N
O
O
R C
N
N
N
NH3
R C
R
+H+,
-FlH-
electron
transfer
R C
COOH
NH2
H2O
R C
-NH4+
O
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
O
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
SCoA
Flox
SCoA
O
O
O
3.71
3.70
Scheme 3.47
Mechanism-based inactivator
Evidence for Radical Intermediates
Electron transfer mechanism for inactivation of
general acyl-CoA dehydrogenase by
(methylenecyclopropyl)acetyl-CoA
B:
H
only pro-R
removed
H
Fl
Fl
SCoA
SCoA
SCoA
*
*
O
*
O
O
Fl
SCoA
O
3.71
Fl
SCoA
O
3.72
Both enantiomers inactivate
very fast—no
stereospecificity
(* is either R- or S)
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
O
SCoA
SCoA
O
O
SCoA
O
O O
HO
FAD
O
O
O
_
_
H+
SCoA
SCoA
O-
O
O
O
O
O
3.73
isolated
Scheme 3.49
Carbanion Followed by Single Electron Mechanism
for General Acyl-CoA Dehydrogenase
H
B
:B
O
SCoA
R
SCoA
R
H H
B:
H
B
N
N
H
O
R
R
N
N
N
NH
N
O
O
SCoA
H
SCoA
R
H
R
R
O
OH
H
B H
N
O
B:
H
O
SCoA
H
H
b
R
N
N
H
B:
N
O
NH
O
O
SCoA
R
H
O
NH
N
O
R
SCoA
R
N
NH
N
O O
a
O
a
NH
N
a
B:
O
R
N
Not in text
Single Electron Transfer Mechanism
Possible mechanisms for monoamine oxidasecatalyzed oxidation of amines
FlH-
Fl
:
+
RCH NH2
3.75
RCH2NH2
3.74
FlHFl
FlH-
Fl
-H
Fl
Fl
X
-H+
Scheme 3.50
RCHNH2
3.77
X
:
•+
RCH2NH2
3.76
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
S
R
N
O
N
S
NH
N
S
H
N
NH
N
H
O
S
O
O
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
R
N
Evidence for Aminyl Radical (radical cation?)
Mechanisms proposed for inactivation of MAO by
1-phenylcyclopropylamine
Fl-
Fl
Fl
14Ph
Fl
NH2
•+
NH2
14Ph
3.79
FlH- S
All products derived
from cyclopropyl
ring opening
+
NH2
Fl
S-
b
Fl+
B
S
t1/2 ~80 min
O
Ph
3.81
3.80
14Ph
a
+
NH2
14Ph
pH 7.2
H2 O
3.85
+
NH2
H
S
Ph
NH2
+
NH
14Ph
2
3.82
14Ph
H2O
3.84
H2O
FlS
1. NaBH4
- H2O
14Ph
3.87
OH
14Ph
2. Raney
Ni
O
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
FlNaB3H4
0.5 N KOH
ca. 1 equiv 3H incorporation
O
14Ph
3.85
14Ph
O
3.83
1. CF3CO3H
2. KOH
14PhOH
Scheme 3.54
Baeyer-Villiger reaction
Inactivation of MAO and Peptide Mapping
CH3
Cys-365
N
H
Ph
Lys-Leu-X-Asp-Leu-Tyr-Ala-Lys
3.88
3.89
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
N
H
-H+
3.88
HO
CH3
Fl-
Fl
Ph
N
H
S
S
NaBH4 O
CH3
H2O
S
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
Ph
NH2
3.90
Fl-
Fl
a
+
NH2
3.91
Ph
+
NH2
3.94
b
FlH-
Ph
Fl
N t Bu
O
Ph
N
3.93
Ph
N
H
3.92
EPR spectrum
(triplet of doublets)
Scheme 3.56
Evidence for -Carbon Radical Intermediate
Oxidation of (aminomethyl)cubane by MAO
Gives product of a cubylcarbinyl radical intermediate
Fl
Fl
NH2
NH2
– H+
a
NH2
3.96
3.95
Fl
Fl
b
–H
FlH–
c
FlH–
CHO
a
NH2
+
NH2
3.98
3.97
detected
Scheme 3.57
further decomposition
and inactivation
Reactions to Differentiate a Radical from
a Carbanion Intermediate
O
R
O
R
A
O
O
R
B
Scheme 3.58
R
Further Evidence for -Carbon Radical with MAO
Mechanism proposed for MAO-catalyzed
oxidation of cinnamylamine-2,3-epoxide
Fl
O
Fl
O
Ph
– H+
Ph
3.99
NH2
Ph
NH2
isolated
HOCH2CHO
+
PhCHO
O
FlH–
H2O
Ph
O
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
O
Ar
14
O
NH2
-H+
Ar
O
O
3.100
3.101
Fl
O
H
3.102
O
O
isolated
Ar
14
Fl
+H+, +H2O
-NH3
evidence for reversible e- transfer
•
•
(Fl  Fl- , Fl -  Fl)
NH2
-14CO2
detected
Ar
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
NHMe
Fl
Fl
ArCxH2O
N y O
N y O
O
O
3.103
-H+
X
NHMe
NHMe
X
ArCxH2O
N y O
3.104
ArCxH2O
O
O
+
NHMe
X
N y O
–
ArCxH2O
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
OH
HO
Mur B
O
HO
O
O
-OOC
NH
O
O
NADPH
H+
UDP
3.105
EP-UDP-GlcNAc
Scheme 3.63
O
NADP+
-OOC
NH
O
O
UDP
3.106
UDP-N-acetylmuramic acid
Hydride Mechanism for a Flavoenzyme
(MurB)
H
R
N
N
O
EP-UDP-GlcNAc
NH
N
H
H
OH
R
N
N
O
-NADP+
-FAD
M+
O
NH
O
H
O
UDP
HO
M+ O
O
O
O
B+
OH
NH
O
O
UDP
HO
M+ O
H
O
O
3.105
O
NH
O
Scheme 3.64
O
OH
R
In situ generation
of FADH
B:
H
NH2
N
O
O
N
O
HO
O
NH
O
O 229Ser
3.106
O
UDP
Evidence for the Hydride Mechanism
MurB-catalyzed reduction of (E)-enolbutyryl-UDPGlcNAc with NADP2H in 2H2O
HO
-OOC
MurB
O
O
CH3
OH
OH
O
NH
O
UDP
NADPD
D2O
O
O
HO
O
O
D
-O
D
3.107
extra Me for
stereochemical
determination
NH
O
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
O
O
HO
O
-O
D
D
H
CH3
Scheme 3.66
O
NH
OH
alkaline
phosphatase
O PO3=
O
O
O
HO
NaOD
O
NH
-O
D O
D
H
CH3
OH
OH
D
-O
D
CH3
3.109
Substrate for D-lactate
dehydrogenase but not
L-lactate dehydrogenase,
therefore 2R stereochemistry
H
Enzymatic Syntheses of (2R,3R)- and (2R,3S)isomers of 2,3-[2H2]hydrobutyrate for NMR
Comparison with 3.109
pyruvate
kinase
O
O-
H3C
D2O
O
H
omit ATP
O
NADD
H3C
OO
D
D-lactate
dehydrogenase
OH
O
(2R, 3R)-2,3-[2H2]-2hydroxybutyrate
O
D
O-
OH
H3C
D2O
pD7
D
D
D
O
pyruvate
kinase
H2O
Scheme 3.67
O
H3C
H
D
O-
D
O
NADD
H3C
D-lactate
H
dehydrogenase
OH
O-
D
O
(2R, 3S)-2,3-[2H2]-2hydroxybutyrate
Stereochemistry of the MurB-catalyzed
Reduction of (E)-enolbutyryl-UDP-GlcNAc
R
N
N
O
HN
M+
HN
B+ H O
RO
O
H
Ser229
N
O
H
O-
N
O
N
O
R
N
reface
B:
M+
H
O-
O
RO
O
H
Ser229
OO
RO
Scheme 3.68
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 H’s; 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)
1Huang,
Z.; Zhang, Q.; Liu, H.-w. Bioorg. Chem. 2003, 31, 494-502.
2Zhang,
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
N
N
NH
N
O
O
S
O
NH
N
O
3.111
O
O
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
O
NH
N
H
O
3.112
compare
structures
R
N
N
H
O2
H2O2
H2O
H
N
N
O
N
H
H
N
O
NH
HO O
3.113
H
N
O
N
H
O
O
NH2
3.114
detected
comes from H2O,
not O2 (using 18O)
H
N
O
NH
O
reduced flavin
Scheme 3.71
Mechanism for an Oxidase-catalyzed Oxidation
of Reduced Flavin to Oxidized Flavin for
Comparison with Urate Oxidase
R
R
N
N
O
N
N
O
a
N
H
B H
NH
NH
N
a
O
O O
B
-H2O2
Flox
H O O
OH
3.53
b
-H2O2
O O
e transfer
radical
combination
R
N
N
3.54
d
NH
N
H
O
O
O O
c
2nd e- transfer
+ H+
H
B
Scheme 3.33
Possible Mechanism for the Urate
Oxidase-catalyzed Oxidation of Urate
H
H
N
N
O
O
probably by
two 1 e- steps
N
O
NH
N
H
H
N
NH
N
O
H
3.112
B:
O
-H2O2
H
N
O
H
O
NH
N
O O
OH
detected
N
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
9
3
8
4
HOOC
7
N
6
5
O
O
3.115
Bound to quinoproteins
Also called methoxatin, coenzyme PQQ
Possible Mechanisms for the Glucose
Dehydrogenase-catalyzed Oxidation of Glucose
COO-
-OOC
Nucleophilic
mechanism
COO-
-OOC
HN
COO-
-OOC
HN
HN
A
-OOC
5
N
Ca2+
4
O
O
from model study
with MeOH C-5
favored over C-4
addition
OH
-OOC
O
H
144His
-OOC
O
O
Ca2+
H
O
..
OH
O
144His
..
H
O
H
HO
N
OH
Ca2+
OH
OH
OH
HO
N
O
144His
H
..
O
O
OH
HO
HO
HO
HO
COO-
-OOC
COO-
-OOC
HN
COO-
-OOC
HN
HN
B
Hydride
mechanism
-OOC
N
5
4
O
Ca2+ H
O
OH
O
OH
HO
HO
-OOC
O
H
144His
..
N
Ca2+
O
O
OH
H
..
-OOC
N
OH
Ca2+
H
O
H
144His
..
O
O
OH
HO
144His
Scheme 3.73
HO
From crystal structure, hydrogen over C-5 carbonyl, suggesting hydride mechanism
Evidence for Nucleophilic Mechanism for Plasma Amine Oxidase
(contains CuII)
Plasma amine oxidase
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
O
O
O
+NH
B+
+NH
3H
isotope
effect
14Ph
H
:B
NaCNB3H3
OH
H
14Ph
H2O
- 3 H+
OH
NH
OH
3H
O
NH3+
NH
14PhCHO
14Ph
1 equiv. 14C
no 3H from
NaCNB3H3
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
O
COOH
N
HN +
1
2
HS
COOH
HS :B
HR
HR
Ar
Scheme 3.75
-O
O
HN +
HR H S
+B
O
HS
HN +
HR
HR
HS
Ar
HS
HS
HR
Ar
HN
HR
Ar
:B
HS
Topa Quinone (TPQ), 6-Hydroxydopa,
is the Actual Cofactor for PAO
O
Leu
Asp
C
CH
NH
Asn
Tyr
CH2
O
1
2
3
5
4
O
OH
3.116
Characterized by Edman degradation, and mass,
UV-vis, resonance Raman, and NMR spectrometries
Using X
NH a Hammett study showed
 = 1.47 ± 0.27 (carbanion-like TS‡)
2
Plasma amine oxidase-catalyzed amine oxidation
with topa quinone shown as the cofactor
CH2
O
O
R
O
NH2
O
O-
:B
H
+
N
H
O-
H
N
H
R
R
OH
3.117 H
B
O
-RCHO
NH2
Scheme 3.76
OH
3.118
O
+
N
H
H2O
OH
R
Model Study for Topa Quinone
O
HN
R
R
NH
R = t-Bu 3.120
i-Pr 3.121
3.122
Et
Me 3.123
O
O
O
O
O
R = OMe
Me
H
O
3.124
3.125
3.126
OH
O
O
OMe
OH
3.119
O
C-5
R = H
OMe
R
3.128
3.129
O
3.127
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
O
O
O
OH
O
OH3 N+
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
OH2
CuII
CH2
CuII
Ph
O
NH2
H2O
H
O
CH2
:B
H O
CuII
H
+
N
O
O-
O-
H
O
CH2
H
O
CHPh
NH
H
OH
CHPh
H2O
PhCHO
NH3
H2O
CuII
CH2
H2 O2
OH2 O
H
+
NH2
O-
Scheme 3.79
CH2
CuII
O2
O
O
H
NH2
OH
3.131
Mechanism Proposed for Reoxidation of
Reduced Topa Quinone
Based on EPR spectroscopy
CuII
H
-2H+
O
O
CH2
CuI
CH2
OH
O2
OH2
O
H
3.132
detected
Scheme 3.80
O
+
NH2
NH
NH2
3.131
CH2
CuII
O
H
OH
H2O2
O-
Mechanism Proposed for Biosynthesis of Topa
Quinone from Tyrosine
-H+
CuII
CuII
CuI
OH
CuI
OH
O
O
O2
CuII
CuII
O
O
O
CuII
O
O
O
H O
O
O
CuII
O
O
O
B:
O
H+
CuII
O2, H+
O
H2O2
O
CuII
CuII
H
O
B:
O
O
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
Asp-Thr-(modified Tyr)-Asn-Ala-Asp
Val-Ala-Glu-Gly-His-(modified Lys)
3.134
Isolated from a proteolytic digestion
Structure of Lysine Tyrosylquinone in
Lysyl Oxidase
Asp-Thr N CHCONH
Val-Ala-Glu-Gly-His
H
NH
CH2
H C
CH2CH2CH2CH2 NH
C
OH
O
O
Lys
3.135
O
Tyr
Asn-Ala-Asp
Enzymes Containing Amino Acid Radicals
Mechanism proposed for galactose oxidase using a covalently bonded
cysteine cross-linked tyrosine radical
Tyr272
O.
H
OH
H
R
Tyr
O . Cu(II)++
Cu(II)++
S
S
Cys228
Cys
3.136
-H+
-HOO-
O.
S
O H
Cu(II)++
S
Cys
H
O2
O2
OH
O
H
R
H
R
H atom transfer
stepwise mechanism
Tyr
S
Cys
O
H
Cys
R
Tyr
Cu(I)+
Cys
O
ER2; radical E2
concerted mechanism
R
Tyr
O . Cu(II)++
Cu(II)+
S
H
H
O
Cys
Scheme 3.82
Tyr
Tyr
O2
S
R
H
-RCHO
Tyr
OH
H
OH Cu(I)+
O
H
R
OH
S
Cys
Cu(II)++
O
H . R
ketyl
radical anion
Mechanism-based Inactivation of Galactose
Oxidase by Hydroxymethylquadricyclane and
Hydroxymethylnorbornadiene
Tyr272
Scheme 3.83
O . Cu(II)++
-B
CH2O-H
Tyr
OH Cu(II)++
S
CH2O
Cys228
3.136
S
Cys
O
C
O
C
H
H
3.137
quadricyclane
analogue
CH2OH
ketyl radicals
O
same as with 3.137
C
3.138
norbornadiene
analogue
Tyr
H
3.139
OH Cu(II)++
S
Cys
inactivated enzyme
complex
[,-2H2] 3.137
kH/kD = 6 on inactivation
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+
O
OCDP
OH
3.140
(PMP)
OH
=O PO
3
O
OH
O
OH
H
N
+
N
3.142
OCDP
OH
3.141
Me
Me
E1
-H2O
OCDP
OH
Pyr
O
H
N
+
Pyr
OCDP
OH
3.143
Pyr = pyridine ring of PMP
NADH, FAD
E1/E3
O
Me
OCDP
OH
3.147
ascarylose
O
NADPH
O
Me
OCDP
OH
3.146
*
Me
Me
HO
Fe(III)Fe(II)S2
O
3.142
O
OCDP
OH
3.145
O
H
N
+
Pyr
OCDP
OH
3.144
Scheme 3.84
Pyridoxamine 5-Phosphate (PMP)
CH2NH2
OH
=O PO
3
N
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
S Cys
S
Fe
Fe
Cys
S
Cys S
Cys S
S
S
Fe
S
S
S Cys
S
Fe
Fe
[2Fe-2S]
Cys
S
Fe
S
Fe
S
Cys S
3.149
3.150
[3Fe-4S]
[4Fe-4S]
1 electron and 2 electron transfers
Cys
S Cys
Fe
S
S
Cys S
3.148
S
Fe
Mechanism Proposed for the Reduction of
CDP-6-deoxy-3,4-glucoseen by E1 and E3
=O
O
PMP
OH
Me
HN +
Me
OCDP
OH
=O
B
B:
H
E1
Me
O
3PO
O- H
=O
H
3PO
Me
HN +
O
N
+
3PO
OH
Me
OCDP
OH
O- H
Me
O
HN +
OCDP
H OH
B
Me
N
+
O
+
N
O H
OCDP
OH
*
FADH-
NAD+
E1, PMP
E3, NADH
**
Fe(III)2S2
E3
NADH
E3
FAD
Fe(III)Fe(II)S2
Fe(III)Fe(II)S2
H+
E1
Fe(III)2S2
FADH
1e- transfer
Fe(III)Fe(II)S2
=O
Me
3PO
O
OCDP
OH
3.145
-PMP
Me
O
=O
+
N
H
3PO
Fe(III)Fe(II)S2
Me
HN +
E3
E1
O
H2O
Fe(III)2S2
Fe(III)2S2
O
OCDP
OH
* In 3H2O, 1 3H in product
** (4R)- and (4S)-[4-3H]NADH both transfer 3H
3H released as 3H O
2
Me
HN +
1e- transfer
Me
+
N
O H
3.151
O
OCDP
OH
EPR evidence
Scheme 3.85
Molybdoenzymes and Tungstoenzymes
OH OH
O
O
HN
H2N
S
H
N
O
O
MoVI
H2N
S
N
H
P
P
O
O-
O
O
H
N
S
N
H
S
N
OPO3=
O
O
HO
N
NH2
NH
N
HN
N
O
O-
O
N
O
HN
3.152
H
N
S
MoVI
O
3.153
O3=PO
O
H
N
S
N
H
N
N
OO
O
N
H
N
S
O-
N
H2N
O
O
O
P
P
O
O
NH
N
NH2
O
OH
OH
NH2
NH
S
O
HN
H2N
N
H
N
N
H
S
O
WVI
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
S
O
O
MoVI
O
S
O
O
S
HN
O
N
OPO3=
N
H HO
H2N
N
S
H
N
O
MoVI
S
OS
O
O
OPO3=
N
H HO
O
H OH
B:
H2 N
IV
S Mo
O
S
O
H
N
N
N
H HO
HN
O
OPO3=
3.152
O from H2O
O
O
O
O
-2e-
HN
H2N
Scheme 3.86
N
H
N
H O
O
IV
S Mo
S
N
H HO
-SO4=
OPO3=
H2N
O-
O
IV
S Mo
O
S
O
H
N
N
N
H HO
HN
S
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
N
H
N
CH3
H
HN
CH3
N
14a
+ H2
Scheme 3.89
H
N
HN
CH3
CH3
N
O
N
3.158
N
H
+
O
H
H2N
+
H+
N
HR H
S
R
pro-R
specific
3.159
R
Model Study for Metal-free Hydrogenase
Reaction of perhydro-3a,6a,9a-triazaphenalene
with tetrafluoroboric acid
N
N
N
+ H+
N
110 °C
N
+
H
strong acid
3.161
antiperiplanar stereoelectronic
effect
Scheme 3.91
N
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)
O
O
-O
H
O
H H
initially, not resonance stabilized
HR
H3C H R
N
N
H
HS
H3C H R
N
ring
H
ring
3.159
Scheme 3.90
+
N
ring
3.160
H
R
H3C
H
ring
H
N
+
N
ring
ring
conformational
change
H + H2
3.158