The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.
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Transcript of The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.
![Page 1: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.](https://reader030.fdocuments.in/reader030/viewer/2022032722/56649ce55503460f949b31d8/html5/thumbnails/1.jpg)
The Organic Chemistry of Enzyme-Catalyzed Reactions
Chapter 3
Reduction and Oxidation
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Redox Without a Coenzyme
Internal redox reaction
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Scheme 3.1
CH3C
O
CH
O
CH3 CHCOOH
OH
3.1 3.2
Reaction Catalyzed by Glyoxalase
methylglyoxal lactic acid
Looks like a Cannizzaro reaction
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Scheme 3.2
Ph C
O
H Ph C
O
H Ph PhCOO- CH2OH
O-
C HPh
HO
Ph C
O
H
+
oxidized reduced
+
HO-
-OH
Cannizzaro Reaction Mechanism
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Scheme 3.3
glutathione
reduced
oxidized
CH3 C
O
C
O
H CH3 C
HO
C
O
SG
H
CH3 C
HO
H
C
O
SG CH3 CHCOO-
OH
glyoxalase I
3.3
+ GSH
3.4
+ GSHglyoxalase II
+ H2O
3.4
Reactions Catalyzed by Glyoxalase I and Glyoxalase II
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Glutathione (GSH)
H3N CHCH2CH2
COO-
CNH
O
CH
CH2SH
C NHCH2CO2-
O(γ-Glu-Cys-Gly)
3.3
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Scheme 3.4
CH3 C
O
C
O
H CH3 C
OH
C
O
SG
H
CH3 C
O
C
O-
SG
CH
H
COO-
OH
CH3
BH
glyoxalase IIGSH +
H SG
B-
H2O
Hydride Mechanism for Glyoxalase
reduced oxidized
Intramolecular Cannizzaro reaction
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• 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
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Scheme 3.5
cis-enediol
CH3 C
O
C
O
H CH3 C
O
C
OH
SG
H
B H
CH3 C
HO
C
O
SG
B+ H
HB:
CH3 C
HO
C
O
SG
H
B:
+ GSH
no exchangewith solvent
3.5
Enediol Mechanism for Glyoxalase
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Scheme 3.6same oxidation state
FCH2C
O
CH
O
C C
HO O
SG
H
FCH2 CH3C C SG
OOglyoxylase
GSH+
3.7
3.83.6
Reaction of Glyoxalase with Fluoromethylglyoxal
Another test for the mechanism
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Scheme 3.7
FCH2C
O
C
O-
H
B+ H
SGC C
HO O
SG
H
CH2F
B:
CH2 C
HO
CSG
O
CH3C C SG
OO
FCH2C
O
CH
O
3.7
3.83.6
GSH
Hydride Mechanism for the Reaction of Glyoxalase with Fluoromethylglyoxal
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Scheme 3.8
FCH2C
O
C
O-
H
B+ H
SG
B:
C C
HO O-
SG
H
CH2F
B+
C C
HO O
SG
H
CH2F
B+
C C
HO O
SG
H
FCH2C C SG
OHO
CH2CH3C C SG
OO
b
a
ab
3.8
3.7
FCH2C
O
CH
O
3.6
GSH
Enediol Mechanism for the Reaction of Glyoxalase with Fluoromethylglyoxal
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Scheme 3.9 F- lossdecreased
FCH2C
O
C
O-
D
B+ H
SG C C
HO O
SG
D
CH2F
B:
CH3C C SG
OO
FCH2C
O
CD
O
3.9
GSH -F-
Hydride Mechanism for the Reaction of Glyoxalase with Deuterated Fluoromethylglyoxal
deuterium isotope effect
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Scheme 3.10 F- lossincreased
Enediol Mechanism for the Reaction of Glyoxalase with Deuterated Fluoromethylglyoxal
deuterium isotope effect
FCH2C
O
C
O-
D
B+ H
SG
B:
C C
HO O-
SG
D
CH2F
B+
C C
HO O
SG
D
CH2F
B+
C C
HO O
SG
D
FCH2C C SG
OHO
CH2CH3C C SG
OO
ba
ab
FCH2C
O
CD
O
3.9
GSH
-F-
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Table 3.1. Comparison of Fluoride Ion Elimination with Fluoromethyl Glyoxal and [1-2H]FluoromethylGlyoxal
Source % Fluoride ion elimination
FCH2
C
O
CH
O
FCH2
C
O
CD
O
yeas t 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
yeas t/D2O 33 .8 ± 0.2 39 .1 ± 0.4
increased F- loss supports enediol mechanism
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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)
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NAD(P)+ NAD(P)H
Enzyme without coenzyme bound - apoenzyme
Enzyme with coenzyme bound - holoenzyme
apoenzyme holoenzymecoenzyme
N
N N
N
NH2
O
HO OH
CH2 OP
O
O-
OP
O
O-
O CH2N
NH2
O
O
OR' HO
N
N N
N
NH2
O
HO OH
CH2 OP
O
O-
OP
O
O-
O CH2N
NH2
O
OOR' HO
HH
3.10a, R' = Hb, R' = PO3
=3.11
Called reconstitution
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Abbreviated Forms
NAD(P)+
(oxidized)NAD(P)H(reduced)
R
N
NH2
O
3.12
R
N
NH2
OHH
3.13
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• 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)
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N
COOHO
OH OH
O3PO
OP2O6-3
N
COOH
O
OH OH
O3PO N
N N
N
NH2
O
HO OH
CH2 OP
O
O-
OP
O
O-
O CH2N
OH
O
O
OH HO
N
N N
N
NH2
O
HO OH
CH2 OP
O
O-
OP
O
O-
O CH2N
NH2
O
O
OH HO
3.14
=
+
3.15
PPi =
3.16
ATP
3.17
PPi
3.18
Gln
ATP
Scheme 3.11
nicotinic acid (vitamin B3) niacin
from ATP
Biosynthesis of Nicotinamide Adenine Dinucleotide (NAD+)
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Figure 3.1
C
H
OH
C
O
C
H
+NH3
C
O
C H
O
C O
O
C C
H H
C C
C N
H H
C N
Reactions Catalyzed by Pyridine Nucleotide-containing Enzymes
Oxidation potential NAD+/NADH is -0.32 V
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Scheme 3.12
In 3H2O, no 3H in NAD(P)H
R C
H
H
O H
N
NH2
O
R
R CO
H
N
NH2
O
R
HH
B: B
H
+
Reactions Catalyzed by Alcohol Dehydrogenases
Mechanism
Hydride mechanism
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Scheme 3.13 No *H found in H2O
Reaction Catalyzed by Alcohol Dehydrogenases Using Labeled Alcohol
R C
O
H N
NH2
OHH
R
+ +
N
NH2
OH
R
*RC H2OH
*
*H2O
Supports hydride mechanism
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Scheme 3.14
3.19
k = 108 s-1
3.20
Cyclopropylcarbinyl Radical Rearrangement
Test for a radical intermediate
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Scheme 3.15
CO2H
O
CO2H
OH
pig heart
lactatedehydrogenase3.21
NADH
Test for the Formation of a Radical Intermediate with Lactate Dehydrogenase
No ring cleavage - evidence against radical mechanism
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Scheme 3.16
Chemical Model for the Potential Formation of a Cyclopropylcarbinyl Radical during the Lactate
Dehydrogenase-catalyzed Reaction
Should have seen ring opening in the enzyme reaction if a cyclopropylcarbinyl radical formed
CO2Me
O
CO2Me
OSnBu3
CO2Me
OSnBu3
CO2Me
O
AIBNΔ
Bu3SnH
Bu3SnH
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Scheme 3.18radical reduction product
Ph CH2Cl
O
Ph CH3
O
3.23 3.24
NADH
Nonenzymatic Reduction of -Chloroacetophenone
Another test for a radical intermediate
Nonenzymatic reaction
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Scheme 3.19hydride reduction product
(stereospecific) X = F, Cl, Br
When X = I, get mixture of 3.25 (X = I) +
Ph CH2X PhX
O OH
*
HLADH
3.25
NADH
Ph CH3
O
(radical reduction product)
Horse Liver Alcohol Dehydrogenase-Catalyzed Reduction of -Haloacetophenones
Supports no radical intermediate
Electron transfer is possible if the reduction potential is low enough
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Stereochemistry
An atom is prochiral if by changing one of its substituents, it changes from achiral to chiral
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Figure 3.2
Stereochemistry:
Determination of the chirality of an isomer of alanine
R,S Nomenclature
H3N COO-
H3C H
A B
C D lowest priority behind
counterclockwise (S)
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Figure 3.3
Caacd Cabcd
CH3 OH
H H
CH3 OH
2H H
chiralprochiral
pro-R hydrogen
prochiral chiral
CH3 OH
H H
CH3 OH
H 2H
chiralprochiral
pro-S hydrogen
R
S
Determination of Prochirality
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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
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Figure 3.4
Determination of Carbonyl and Alkene (sp2) Chirality
CH3C
O
H CH3C
CH2
H
si face
re face
si face
re face
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Scheme 3.20
H
R
NH2
O
N N
H
NH2
D
R
O
D
R
NH2
O
N N
D
NH2
H
R
O
+
3.26
+ CH3CDO
+
3.27
+ CH3CHO
CH3CD2OH
CH3CH2OH
A
B
YADH
YADH
Reaction of Yeast Alcohol Dehydrogenase (YADH) with (A) [1,1-2H2]ethanol and NAD+
and (B) Ethanol and [4-2H]NAD+
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Scheme 3.21
No 2H
No H
stereospecificH
R
NH2
O
N
CH3COH
H
D
D
R
NH2
O
N
H
R
NH2
O
N
+ CH3CHO
+
3.28 3.26
N
H
NH2
D
R
O
+
3.26
N
D
NH2
H
R
O
+
3.28
+ CH3CHO
+
3.27
YADH
YADHCH3CH2OH
YADHCH3CHO
A
B
C
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+
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only one H is transferred
re-face
N
R
NH2
OHRHS
NR
HR
HS
H2N
O
3.29
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Not all enzymes transfer the same hydride
Scheme 3.22
pro-R
pro-S transferred
(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
CH3CD2OH
N
R
DH
NH2
O
H2C CH C OP
O
OHOP N
R
NH2
OD
H2C CH CHO
OHOP
+ CH3CDO
3.26
+ NAD+
G3PDH
3.30+
+ + + Pi
3.26
A
B
YADH
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Figure 3.5
Transition State for Hydride Transfer
syn-axial electrons assist
Anti- and syn- conformations of NADH
HS HR
HS
N N
OHH
O
OH OHH
O
OH
RO RO
anti conformation syn conformation
:
pro-Rtransfer
pro-Stransfer
O
H2N
O
HR
NH2
:
Boat-like TS‡
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Figure 3.6
The enzyme may drive equilibriumBoat-boat equilibria of NADH
N
HR
CONH2
HS
ON
HR
HS
O
CONH2
N
HS
HR
ON
HS
HR
O
H2NOC
H2NOC
OHHO
RO RO
OHHO
RO
HO OHHO OH
RO
anti-NADH
HR transfer
syn-NADH
HS transfer
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Oxidation of Amino Acids to Keto Acids
Scheme 3.24
+N
CONH2
R
CO2-
CO2-
NH2H
N
CONH2
R
CO2-
COO-
HH
NH2
OH
CO2-
CO2-
O NH3
CO2-
CO2-
NH3O
H
H
+ H
D165
D165
..
H3N K113
H3N K89
H3N K89
H3N K113
NH2K125NH2K125
H OOC
NADPH
+
D165
NH3K125
H3N K113
H3N K89
..
D165
H3N K113
H3N K89
+
NH3K125HOOC -OOC
-OOC
Possible mechanism for the reaction catalyzed by glutamate dehydrogenase
Hydride transfer
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Scheme 3.25
Oxidation of Aldehydes to Carboxylic Acids
covalent catalysis
via hydrate
(A) Covalent catalytic mechanism for the oxidation of aldehydes by aldehyde dehydrogenases; (B) noncovalent
catalytic mechanism for the oxidation of aldehydes by aldehyde dehydrogenases
O
R H
B H–S
B:
R H
OS
HO
R S
B:
R H
OOH OH
R OH
++ NADH
O
R OH
RCHO + H2O + NADH
3.31
O HH
3.32
B–
3.33
NAD+
A
B
NAD+
Hydride transfers
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Scheme 3.27
Oxidation of Deoxypurines to Purines
inosine MP
xanthine MP
Mechanism for the oxidation of inosine 5-monophosphate by inosine 5-monophosphate dehydrogenase
HN
N N
N
OH B+
B:
RP
HN
N N
N
OH :B
RPX
H
N+
NH2
O
R
HN
N N
N
OH B+
XRP
N
NH2
O
R
B
H H
HN
N N
N
O
X RP
H :B
OH
B:
H
B+HB+
HN
N N
N
O
X H
B:
RPO
X H
H OH
3.36
3.37
H
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Scheme 3.28
N
HN COOH
N
HN COOH
OH3.39
urocanase
3.40
D
D
D2O
An Atypical Use of NAD+
Reaction catalyzed by urocanase
NAD+ in a Nonredox Reaction
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“substrate”
exchangeable proton
apo-urocanase reconstituted with [13C]NAD+
Urocanase Reaction Run with a [13C] Pseudo-substrate
N+
NH2
O
R3.41 3.42
N
HN COOH
reducedside chain
13
H
13
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NMR determined
N
NH2
O
R
N
HN COO-
13
13
3.43
Adduct Isolated after Chemical Oxidation
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N+
NH2
O
R
H
B
H
N
N
HCOO-
N
NH2
O
R
N
N+
H
COO-
N
NH2
O
R
N
N
H
COO-
B+H
NNH2
O
R
N
+N
H
COO-
B:
H
OHH
N
OH
NNH2
O
R
N
N+
HCOO-
N
HCOO-
OH
oxidative quench oxidizes this reduced adduct
When 3.41 is used, the reaction stops here.
:B
H
H
+ NAD+
Scheme 3.29
exchangeable
solvent incorporated
Mechanism Proposed for Urocanase
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Scheme 3.31
Flavin Coenzymes
riboflavin (vitamin B2)
FMN FAD
Biosynthetic conversion of riboflavin to FMN and FAD
6N
N
NH
N O
CH2
(CHOH)3
CH2OH
O
CH2
(CHOH)3
CH2O P
O
O-
O-
CH2
(CHOH)3
CH2O P
O
O
O-
P
O
O-
O CH2O
HO OH
N
N
N
N
NH2
5
8
7
ATP
N
N
NH
N O
O
9
1010a
4a
ADP PPi
N
N
NH
N O
O3.48
8a
3.49 3.50
ATP
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Scheme 3.32
oxidized semiquinone reduced
some covalently attached to The protein at these positions
Interconversion of the Three Oxidation States of Flavins
N
N
NH
N O
R
O
N
N
NH
N O
R
O
NH
N
NH
N O
R
O3.52
_
FlH
(Fl)
+1e-
-1e--1e-
+1e-
Fl
3.51
N
N
N
N
O
O
R
N
N N
NO
O
R
H
H
H
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Figure 3.8
C
H
OH
C
O
C
H
NH2
C
O
+
CH2 CH2 C
O
CH CH C
O
HS SH S S
NAD+
NH4+
NADH
Redox Reactions Catalyzed by Flavin-dependent Enzymes
![Page 50: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.](https://reader030.fdocuments.in/reader030/viewer/2022032722/56649ce55503460f949b31d8/html5/thumbnails/50.jpg)
Scheme 3.33
only if spin inversion occurs
Oxidases vs. DehydrogenasesMechanisms for an oxidase-catalyzed oxidation of
reduced flavin to oxidized flavin
Oxidases use O2 for reoxidation of reduced flavin coenzyme
NH
N
NH
N
O
O
R
N
N
NH
N O
OH O
OH
R
B H O O B
O O
N
N
NH
N O
OH
R
BHO O
2nd e- transfer + H+
3.53
3.54
e- transferb
a
a
radical combination
Flox
c
d
-H2O2
-H2O2
b
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Scheme 3.34
NH
N
NH
N
O
O
R
N
N
NH
N
O
O
R
N
N
NH
N
O
O
R
HB AcceptorAcceptor
Acceptor
Mechanism for a dehydrogenase-catalyzed oxidation of reduced flavin to oxidized flavin
Dehydrogenases Use Electron Transfer Proteins to Reoxidize Reduced Flavin
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Scheme 3.35
Substrate + Enzyme-Flox Oxidized substrate (product)
+ Enzyme-FlH-
Enzyme-FlH- + Acceptor (O2)
Enzyme-Flox + Reduced acceptor (H2O2)
Mechanisms for Flavoenzymes
Overall reaction of flavoenzymes
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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
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Two-Electon Mechanism (Carbanion)
D-Amino acid oxidase (DAAO) catalyzes the oxidation of D-amino acids to -keto acids and ammonia
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Scheme 3.36
Evidence for MechanismIonization of substituted benzoic acids
Hammett Study
KaCO2H + H2O
XCO2
- + H3O+
X
As X becomes electron withdrawing, equilibrium constant (Ka) should increase
Derivation of the Hammett Equation
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Scheme 3.37
Reaction of hydroxide ion with ethyl-substituted benzoates
kCO2Et + HO-
XCO2
- + EtOHX
A Similar Relationship Should Exist for a Rate Constant (k) where Charge Develops in the Transition State
As X becomes electron withdrawing, rate constant (k) should increase
![Page 57: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.](https://reader030.fdocuments.in/reader030/viewer/2022032722/56649ce55503460f949b31d8/html5/thumbnails/57.jpg)
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
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Figure 3.9
Linear Free Energy RelationshipExample of a Hammett plot
p-OCH3
p-CH3
m-CH3
p-F
m-F
p-Cl
m-Cl
p-NO2
m-NO2
o-CH3
o-Fo-Cl
o-NO2
log 105 Ka
1.0 2.0 3.0
1.0
2.0
3.0
4.0
5.0
p-NH2
H
Ortho-substituent points are badly scattered because of steric interactions and polar effects
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log k/k0 = log K/K0 (3.3)
log k/k0 = (3.4)
reaction constant
electronic parameter (substituent constant)
- slope carbocation mechanism+ slope carbanion mechanism
EWG +EDG -
Hammett Relationship (Equation)
depends on type of reaction and reaction conditions
depends on electronic properties of X
H = 0
![Page 60: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.](https://reader030.fdocuments.in/reader030/viewer/2022032722/56649ce55503460f949b31d8/html5/thumbnails/60.jpg)
= +5.44 = +0.73
X = EWG, Vmax
carbanionic TS‡
C
H
NH3+
COOH
3.55
C
H
NH3+
COOH
3.56
CH2
X X
Application of Hammett Equation to Study of an Enzyme Mechanism
D-Amino acid oxidase
Effect of X diminished by -CH2-
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Scheme 3.38
C
H
NH3+
COOH C
NH3+
COOH C
NH
COOH
3.55
X X X
Proposed Intermediate in the D-amino Acid Oxidase-catalyzed Oxidation of
Substituted Phenylglycines
What is the function of the flavin?
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Scheme 3.39
exclusive (in N2)
exclusive (in O2)
40 : 60 (in air)
Further Evidence for a Carbanion IntermediateDAAO-catalyzed oxidation of -chloroalanine
under oxygen and under nitrogen
Total amount of product(s) is the same under all conditions
H2C C
Cl
H
NH3
COO-
:B Enz Fl
H2C C
Cl
H3C
NH3+
COO-
C COO-
NH2
H2C C COO-
NH3+
H2C
Cl
C
NH2
COO-
100% N2
H2C
Cl
C
O
COO-
irreversible100% O2
reversible
Enz-Fl +
3.57
+ Enz-FlH2
H3C C COO-
O
Enz-Fl
3.593.60
3.58
-Cl-
H2O
O2
H2O
H2O2
+
+
expected eliminationproduct
![Page 63: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.](https://reader030.fdocuments.in/reader030/viewer/2022032722/56649ce55503460f949b31d8/html5/thumbnails/63.jpg)
Scheme 3.40
No adduct detected enzymatically
N
N
NCH3
N
Et
O
O
N
N
NCH3
N
Et
O
ONH
CH2Ph
CH3CH3
PhCH2NH2
CH3CN
Where on the flavin does the nucleophilic attack occur?
Evidence against C4a addition
Nonenzymatic reaction of benzylamine with N5-ethylflavin
![Page 64: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.](https://reader030.fdocuments.in/reader030/viewer/2022032722/56649ce55503460f949b31d8/html5/thumbnails/64.jpg)
Scheme 3.41
detected in absence of AMP
Evidence for N5 Addition
Reverse reaction catalyzed by AMP-sulfate reductase
N
N
NH
N O
R
O
N
N
NH
HN O
R
OSO3
=
N
N
NH
N O
R
O
H
H: SO3
=
AMP-SO3=
in the presenceof AMP
+
3.61
+H+
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Scheme 3.425-deazaflavin
Initial Evidence for N5 Attack and for Two-electron Chemistry
N
NH
N O
R
O
NR
HH
NH2
O
N
NH
HN O
R
OH H
NR
NH2
O
variousflavoenzymes
3.62
+
+
H
+
NADH-dependent reduction of 5-deazaflavin by various flavoenzymes
![Page 66: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.](https://reader030.fdocuments.in/reader030/viewer/2022032722/56649ce55503460f949b31d8/html5/thumbnails/66.jpg)
Figure 3.10
Inappropriate flavin substitute
N
H H
N
NH
HN O
R
OH H
O
NH2
Reduced5-deazaflavin
R
NAD(P)H
Comparison of Reduced 5-Deazaflavin with Reduced Nicotinamide
Favors 2-electron reactions because of resemblance to NADH
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Inverse 2° deuterium isotope effect; therefore sp2 sp3 in TS‡, consistent with conversion to carbanion and nucleophilic addition
3.63
NH
H3C
O
ON
H
H3C
O
O
Support for Covalent Carbanionic Mechanism with DAAO rather than
Electron Transfer Mechanism
![Page 68: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.](https://reader030.fdocuments.in/reader030/viewer/2022032722/56649ce55503460f949b31d8/html5/thumbnails/68.jpg)
B:
H
C
NH3
R COOH C
NH3
R COOH
N
N
NH
N O
R
ON
N
NH
N O
R
O
C
NH2
R COOH
C
NH2
R COOH C
O
R COOH
a
N
N
NH
N O
R
O
a
C
NH2
R COOH
b
b :
:
c
d
+H+, -FlH-
radicalcombination
electrontransfer
+H+, -FlH-
H2O
-NH4+
-H+
Scheme 3.43
No base in crystal structure, but -H in line with flavin Not clear how proton is removed
Covalent Carbanion versus Radical Mechanisms for DAAO (Hammett study suggested carbanionic)
favored
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Scheme 3.46
R
O
SCoA R
O
SCoA
Fl FlH-
3.68 3.69
Carbanion Mechanism Followed by 2 One-electron Transfers
Reaction catalyzed by general acyl-CoA dehydrogenase
![Page 70: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.](https://reader030.fdocuments.in/reader030/viewer/2022032722/56649ce55503460f949b31d8/html5/thumbnails/70.jpg)
Scheme 3.47
3.70
SCoA
O
B:
H
SCoA
O
SCoA
O
FlH-
Flox
3.71
Initial Mechanism Proposed for Mechanism-based Inactivation of General Acyl-CoA Dehydrogenase by
(Methylenecyclopropyl)acetyl-CoA
Mechanism-based inactivator
![Page 71: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.](https://reader030.fdocuments.in/reader030/viewer/2022032722/56649ce55503460f949b31d8/html5/thumbnails/71.jpg)
Scheme 3.48
Evidence for Radical Intermediates
only pro-R removed
Both enantiomers inactivate
Electron transfer mechanism for inactivation of general acyl-CoA dehydrogenase by (methylenecyclopropyl)acetyl-CoA
SCoA
O
B:
H
SCoA
O
SCoA
O
SCoA
O
SCoA
O
Fl
Fl
Fl
Fl
very fast—nostereospecificity(* is either R- or S)
* *
H
*
3.723.71
consistent with a radical pathway
![Page 72: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.](https://reader030.fdocuments.in/reader030/viewer/2022032722/56649ce55503460f949b31d8/html5/thumbnails/72.jpg)
Scheme 3.49
Other Evidence for Radical Intermediate
isolated
Mechanism proposed for formation of 3.73 during oxidation of (methylenecyclopropyl)acetyl-CoA by
general acyl-CoA dehydrogenase
SCoA
O
SCoA
OO O
SCoA
OOO
SCoA
OO O
SCoA
OOO-
FAD
SCoA
OO
HO _
_
3.73
3.72
FADO2
H+
![Page 73: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.](https://reader030.fdocuments.in/reader030/viewer/2022032722/56649ce55503460f949b31d8/html5/thumbnails/73.jpg)
Carbanion Followed by Single Electron Mechanism for General Acyl-CoA Dehydrogenase
N
N
NH
N O
R
ON
N
NH
N O
R
O
R
O
SCoA
H HB:
H B
R
OH
SCoA
H
B
:B
H
R
O
SCoA
H
R
O
SCoA
HR
O
SCoA
H
HB:
N
N
NH
N O
R
O
R
O
SCoA
H
aa
a
b
B HN
N
NH
N O
R
OH
N
N
NH
N O
R
O
R
O
SCoA
H
HB:
B:
Not in text
![Page 74: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.](https://reader030.fdocuments.in/reader030/viewer/2022032722/56649ce55503460f949b31d8/html5/thumbnails/74.jpg)
Scheme 3.50
Single Electron Transfer Mechanism
either Fl or amino acid residue
-•
Possible mechanisms for monoamine oxidase-catalyzed oxidation of amines
RCH NH2
XX
NH2R
FlFl
FlH-
•+
Fl
+FlH-Fl
3.74 3.75
FlH-
3.76 3.77 3.78
RCHNH2-H+
RCH2NH2
RCH2NH2
-H
![Page 75: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.](https://reader030.fdocuments.in/reader030/viewer/2022032722/56649ce55503460f949b31d8/html5/thumbnails/75.jpg)
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.
Mechanism Proposed for Generation of an Active-site Amino Acid Radical during Monoamine
Oxidase-catalyzed Oxidation of Amines
N
N
NH
N O
R
OS
H
S
NH
N
NH
N O
R
OS
S
![Page 76: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.](https://reader030.fdocuments.in/reader030/viewer/2022032722/56649ce55503460f949b31d8/html5/thumbnails/76.jpg)
Scheme 3.52
Cyclopropylaminyl Radical Rearrangement
NR NR
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Scheme 3.53
Evidence for Aminyl Radical (radical cation?)Mechanisms proposed for inactivation of MAO by
1-phenylcyclopropylamine
NH214Ph NH2
14Ph 14Ph NH2
FlH- S
Fl-
NH214Ph
Fl-
O14Ph
S
NH214Ph
14PhO
S
O14Ph
14Ph
OH
14Ph
pH 7.2
t1/2 ~80 min
Fl+
+
+
1. NaBH4
2. Raney Ni
- H2O
Fl
Fl3.79 3.80 3.81
3.82
3.83
3.843.85
3.863.87
a
b
•+
H2OH2O
H2O
Fl-Fl
Ph NH2
+
S-
S
NH2Ph
B+
H
All products derived from cyclopropyl ring opening
![Page 78: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.](https://reader030.fdocuments.in/reader030/viewer/2022032722/56649ce55503460f949b31d8/html5/thumbnails/78.jpg)
Scheme 3.54
Chemical Reactions to Characterize the Structure of the Flavin Adduct Formed on Inactivation of
MAO by 1-Phenylcyclopropylamine
Fl-
O14Ph
3.83
ca. 1 equiv 3H incorporation
1. CF3CO3H
O14Ph
14PhOH
0.5 N KOH
3.85
2. KOH
NaB3H4
Baeyer-Villiger reaction
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Cys-365
Inactivation of MAO and Peptide Mapping
MALDI-TOF gives mass corresponding to X as
3.88
Ph NH
CH3
3.89
Lys-Leu-X-Asp-Leu-Tyr-Ala-Lys
HO S
Cys
![Page 80: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.](https://reader030.fdocuments.in/reader030/viewer/2022032722/56649ce55503460f949b31d8/html5/thumbnails/80.jpg)
Scheme 3.55 (modified)
Mechanism Proposed for Inactivation of MAO by N-cyclopropyl--methylbenzylamine
Ph
CH3
NH Ph
CH3
NH
S
SO
SHO
3.88
Ph
CH3
NH
Ph
CH3
NH
Ph
CH3
NH2
Fl Fl
H2ONaBH4
Fl-Fl
S
Ph
CH3
NH
+H+
-H+
![Page 81: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.](https://reader030.fdocuments.in/reader030/viewer/2022032722/56649ce55503460f949b31d8/html5/thumbnails/81.jpg)
Scheme 3.56
Further Evidence for Aminyl Radical (radical cation?) Intermediate
Mechanism proposed for MAO-catalyzed oxidation of 1-phenylcyclobutylamine and
inactivation of the enzyme
NH2Ph NH2Ph Ph NH2
t
NH2Ph
NHPhNPh
PhN
Fl-
BuFl
Fl
Fl
EPR spectrum(triplet of doublets)
FlH-
++
Fl
3.91
3.923.93
3.94
O
3.90
a
b
b
![Page 82: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.](https://reader030.fdocuments.in/reader030/viewer/2022032722/56649ce55503460f949b31d8/html5/thumbnails/82.jpg)
Scheme 3.57
Evidence for -Carbon Radical IntermediateOxidation of (aminomethyl)cubane by MAO
NH2 NH2 NH2
NH2
NH2
CHO
FlH–– H
– H+Fl
+
Fl
Fl
3.95
a
b
3.96
FlFlH–
a
c
3.97
3.98
further decompositionand inactivation
detected
Gives product of a cubylcarbinyl radical intermediate
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Scheme 3.58
Reactions to Differentiate a Radical from a Carbanion Intermediate
O
OR
R
O
RO
R
A
B
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Scheme 3.59
Further Evidence for -Carbon Radical with MAO
Mechanism proposed for MAO-catalyzed oxidation of cinnamylamine-2,3-epoxide
Ph
NH2
O
Ph
NH2
OPh
NH2
O
OPhNH2
Ph ONH2
Fl Fl
– H+
FlH– Fl
3.99
+H2O
PhCHO
HOCH2CHO
isolated
No products of a two-electron epoxide ring opening detected
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Scheme 3.60
More Evidence for -Carbon Radical
evidence for reversible e- transfer (Fl Fl , Fl Fl)-• -•
Mechanism proposed for MAO-catalyzed decarboxylation of cis- and trans-5-(aminomethyl)-3-
(4-methoxyphenyl)-2-[14C]dihydrofuran-2(3H)-one
O
O
Ar
NH2
3.101a
14 O
O
Ar
NH3
14
3.100
-14CO2
O
O
Ar
NH2Fl Fl
3.101
Ar
NH2
14
FlFl
+H+, +H2O -NH3
Ar
O
H
3.102
-H+
isolated
detected
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Scheme 3.61
Evidence for a Covalent Intermediate
When x = 3 and y = 14, both radiolabels are incorporated into the protein
Mechanism proposed for inactivation of MAO by (R)- or (S)-3-[3H]aryl-5-(methylaminomethyl)-2-oxazolidinone
–
Fl Fl
Fl
FlH
+
–
N O
NHMe
O O
NHMe
N OArCxH2O
X
X
X
ArCxH2O y
3.103
3.104
y
N O
NHMe
O
ArCxH2O yN O
NHMe
O
ArCxH2O y
N O
NHMe
O
ArCxH2O y
-H+
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Example of a Hydride Mechanism
Scheme 3.63
UDP-N-acetylmuramic acid
Reaction catalyzed by UDP-N-acetylenolpyruvylglucosamine reductase (MurB)
2nd step in bacterial peptidoglycan biosynthesis
O
OH
ONH
HO
O UDP
O-OOC
O
OH
ONH
HO
O UDP
O-OOC
3.106
Mur B
NADPH NADP+
3.105
H+
EP-UDP-GlcNAc
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Scheme 3.64
Hydride Mechanism for a Flavoenzyme (MurB)
RN
N
N
NH
N
O
NH2
OH H
R
RN
N
N
NH
OH
B+ H
O O
O
OH
ONH
HO
O UDP
OO
OM+
B:
O
OH
ONH
HO
O UDP
OO
O
3.106
M+
EP-UDP-GlcNAc
H
H O
-NADP+
229Ser
3.105
O
OH
ONH
HO
O UDP
OO
OM+
-FAD
In situ generationof FADH
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Scheme 3.65
Evidence for the Hydride Mechanism
extra Me for stereochemical determination
anti-addition
A radical mechanism is not expected to be stereospecific
MurB-catalyzed reduction of (E)-enolbutyryl-UDP-GlcNAc with NADP2H in 2H2O
OHO
O
OH
O UDPNHO
-OOC
CH3
OHO
O
OH
O UDPNHO-O
O
H
D
CH3
D
MurB
NADPDD2O
3.1073.108
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Scheme 3.66
Determination of the Stereochemistry of 3.108
D-configuration
Substrate for D-lactate dehydrogenase but not L-lactate dehydrogenase,therefore 2R stereochemistry
Conversion to 2-hydroxybutyrate of the product formed from MurB-catalyzed reduction of (E)-enolbutyryl-UDP-GlcNAc with NADP2H in 2H2O
3.108
alkalinephosphatase OH
-O
O
H
D
CH3
D
OHO
O
OH
O PO3=
NHO-O
O
H
D
CH3
D
OHO
O
OH
OHNH
O-O
O
H
D
CH3
D
3.109
NaOD NaOD
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Scheme 3.67
omit ATP
Enzymatic Syntheses of (2R,3R)- and (2R,3S)-isomers of 2,3-[2H2]hydrobutyrate for NMR
Comparison with 3.109
O
O-
O
pyruvatekinase H3C
OO-
OHD D-lactatedehydrogenase
H3CO-
OHD
D OH
(2R, 3R)-2,3-[2H2]-2- hydroxybutyratepD7
pyruvatekinaseH3C
OO-
ODD
H3CO
O-
ODH
H3CO-
ODH
D OH
(2R, 3S)-2,3-[2H2]-2- hydroxybutyrate
D-lactatedehydrogenase
D2O
NADD
D2O
H2O
NADD
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Scheme 3.68
re-face
Stereochemistry of the MurB-catalyzed Reduction of (E)-enolbutyryl-UDP-GlcNAc
N
HN
N
N
O
O
R
H
O-
O
RO
M+
H
Ser229
OH
N
HN
N
N
O
O
R
O-
O
RO
M+B: H
Ser229
OH
O-
ORO
H
B+
R
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Scheme 3.69
D isotope effects on both H’s; therefore concerted
Reaction Catalyzed by Dihydroorotate Dehydrogenase
HN
NH
O
O
H
COOH
H
H
Fl
HN
NH
O
O COOH
3.110
FlH-+
:B
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Unusual Reaction Catalyzed by a FlavoenzymeUDP-galactopyranose mutase (UGM)
Requires FAD; only reduced enzyme is active
Absorption spectrum characteristic of N5-monoalkylated flavin
When UGM was incubated with UDP-[3H]-galactopyranose and treated withNaCNBH3, 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
pKa of N5 of reduced FAD is 6.7, suggesting can be deprotonated
Mass spectrum consistent with a flavin-galactose adduct
Soltero-Higgin, M.; Carlson, E. E.; Gruber, T. D.; Kiessling, L. I. Nature Struct. Mol. Biol. 2004, 11, 539-543
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2- and 3-F UDP-galactopyranose are substrates; excludes a mechanism involving oxidation at C2 or C3.2
2Zhang, Q.; Liu, H.-w. J. Am. Chem. Soc. 2001, 123, 6756-6766.
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.
UGM reconstituted with 5-deazaFAD is inactive.1
UDP-galactopyranose mutase (UGM)
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Mechanism of UDP-galactopyranose mutase (UGM)
Mansoorabadi, S. O.; Thibodeaux C. J.; Liu, H.-w. J. Org. Chem.. 2007, 72, 6329-6342.
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Artificial Enzyme (Synzyme)
Scheme 3.70
papaincatalyzes oxidation of NADH to NAD+
Synthesis of flavopapain
N
N
NH
N
Br
Me
O
OO
S-
N
N
NH
N
Me
O
OO
S
3.111
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Scheme 3.71
No flavin, but substrate reacts like a flavin
detected
comes from H2O, not O2 (using 18O)
Unusual Reaction Catalyzed by Urate Oxidase
NH
HN O
O
N
NH
R
reduced flavin
NH
HN O
O
HN
NH
ONH
N O
O
HN
NH
O
HO
NH2
HN
3.112
OHN
NH
3.114
O
3.113
O
O2 H2O2
H2O
compare structures
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Scheme 3.33
Mechanism for an Oxidase-catalyzed Oxidation of Reduced Flavin to Oxidized Flavin for
Comparison with Urate Oxidase
NH
N
NH
N
O
O
R
N
N
NH
N O
OH O
OH
R
B H O O B
O O
N
N
NH
N O
OH
R
BHO O
2nd e- transfer + H+
3.53
3.54
e- transferb
a
a
radical combination
Flox
c
d
-H2O2
-H2O2
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Scheme 3.72
detected
Just like mechanism for oxidation of reduced flavin by O2
Possible Mechanism for the Urate Oxidase-catalyzed Oxidation of Urate
NH
N O
O
HN
NH
ONH
N O
O
HN
NO
O
H
OH
H
B:
NH
N O
O
HN
NO
3.112 H OH
probably bytwo 1 e-
steps
B:
3.113-H2O2
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Pyrroloquinoline Quinone Coenzymes (PQQ)
Bound to quinoproteins
N
HN
HOOC O
O
HOOC
COOH
3.115
2
3
4
56
7
8
9
1
Also called methoxatin, coenzyme PQQ
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Scheme 3.73
Nucleophilic mechanism
from model study with MeOH C-5 favored over C-4 addition
Hydride mechanism
Possible Mechanisms for the Glucose Dehydrogenase-catalyzed Oxidation of Glucose
N
HN
-OOC O
O
-OOCCOO-
54
Ca2+ 144His..
N
HN
-OOC OO
-OOCCOO-
Ca2+
O
O
OH
HO HO
OHH
N
HN
-OOC OH
O
-OOCCOO-
Ca2+
OO
OH
HO HO
OH
A
B
N
HN
-OOC O
O
-OOCCOO-
54
Ca2+ 144His..
OOH
HO HO
OHH
O
O
O
OH
HO HO
OHH
H
H
N
HN
-OOC O
O
-OOCCOO-
Ca2+ H
OO
OH
HO HO
OH
H
H
144His..
H
N
HN
-OOC OH
O
-OOCCOO-
Ca2+ H
144His..
144His..
144His..
From crystal structure, hydrogen over C-5 carbonyl, suggesting hydride mechanism
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O
O
14Ph NH2
+NH
O
H
14Ph
:B
B+H +NH
OH
14Ph
NH
O
14Ph
3HNH
OH
14Ph
NH3+
OH
NH2 HN
+
14Ph
H2N14PhCHO
14Ph
+3H
+
-3H+
NaCNB3H3
H2O
NaCNB3H3
Scheme 3.74
Evidence for Nucleophilic Mechanism for Plasma Amine Oxidase
originally thought it was a PQQ enzyme (We will see it is not)
3H isotope effect
1 equiv. 14C no 3H from NaCNB3H3
Therefore excludes oxidation to 14PhCHO followed by Schiff base formation with a Lys
Schiff base mechanism proposed -- NaCNBH3 inactivates the enzyme in the presence of substrate
Plasma amine oxidase (contains CuII)
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Isotope Labeling Shows Syn Hydrogens are Removed (one-base mechanism)
Scheme 3.75
PQQ is not the actual cofactor for PAO
Stereochemistry of the reaction catalyzed by plasma amine oxidase (PAO)
N
NH
COOH
O
HN
COOH
COOH
:BHS
HR
Ar
HR
HS
HR
HR
-O
HN
Ar
+B
HS
HS
OHS HN HR
HR
ArHS
:B
OHS HN
HR
HS
Ar
+ ++
12
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Characterized by Edman degradation, and mass, UV-vis, resonance Raman, and NMR spectrometries
OH
O
O
CH2
CH C
O
AspNH TyrAsnLeu
3.116
12
3
45
Topa Quinone (TPQ), 6-Hydroxydopa, is the Actual Cofactor for PAO
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Using a Hammett study showed
= 1.47 ± 0.27
Plasma amine oxidase-catalyzed amine oxidation with topa quinone shown as the cofactor
Scheme 3.76
NH2X
O
O
O-
CH2 O
NH
O- R
H
H
:BO
NH
OH R
O
NH
OH R
O
NH2
OH
R NH2
+
+
3.117B
3.118
H
-RCHO
H2O
(carbanion-like TS‡)
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NHHN
O
O
OH
O
O
O
O
OH
R
O
O
R
3.119
O
O
OMe
O
R
O
R = R = OMeMeH
3.1243.1253.126
R = HOMe
3.1283.129
3.127
t-Bui-PrEtMe
3.1203.1213.1223.123
C-5
Preferential attack at C-5 carbonyl by nucleophiles
Model Study for Topa Quinone
Resonance Raman spectrum shows carbonyl at C-5 has greater double bond character (more reactive) than at C-2 or C-4
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Scheme 3.77 Deactivates C-2 and C-4 carbonyls, so C-5 carbonyl is more reactive
Chemical Model Study for the Mechanism of Topa Quinone-dependent Enzymes
O
O
OH
NH2 O
O
O-
H3N+
O
O
O
H3N+
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Scheme 3.79
Mechanism for Plasma Amine Oxidase
Detailed Mechanism Proposed for Topa Quinone-dependent Enzymes
O
CH2
OO-
Ph NH2 O
CH2
N
O-H
H
:B
O
CH2
NH2
O-
O
CH2
NH
OH CHPh
+
3.131
OH2
OH H O
H H
O
CH2
NH2
OH
OH HOH2
+
CHPh
CuII H2O2 O2
PhCHO
H2O
CuII
H2ONH3
H2OCuII
CuII
CuII
![Page 110: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.](https://reader030.fdocuments.in/reader030/viewer/2022032722/56649ce55503460f949b31d8/html5/thumbnails/110.jpg)
Scheme 3.80
Based on EPR spectroscopy
detected
Mechanism Proposed for Reoxidation of Reduced Topa Quinone
O
CH2
NH2
O-
3.1323.131
O
CH2
NH2
OH
OH H OH2
+
O
CH2
NH
O
OH
H
-2H+
CuICuIIH2O2O2CuII
![Page 111: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 3 Reduction and Oxidation.](https://reader030.fdocuments.in/reader030/viewer/2022032722/56649ce55503460f949b31d8/html5/thumbnails/111.jpg)
Scheme 3.81
Mechanism Proposed for Biosynthesis of Topa Quinone from Tyrosine
Topa quinone is ubiquitous - found in bacteria, yeast, plants, mammals
OH OH
CuII
O
CuI
O
CuI
O
CuII
OOO
CuII
OO
H
B:
O
CuII
OO
O
CuII
OO
O
CuIIO
OH
B:O
CuIIO
OH
O
CuII
O
O
O2, H+
TPQ
CuII -H+
O2
H2O2
H+
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in methylamine dehydrogenase
Hammett study with +
3.133
NH
NH
O
O
ProteinProtein
Tryptophan Tryptophylquinone Coenzyme
Observed by X-ray analysis
NH2X
(carbanion mechanism)
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Isolated from a proteolytic digestion
3.134
Asp-Thr-(modified Tyr)-Asn-Ala-Asp
Val-Ala-Glu-Gly-His-(modified Lys)
Coenzyme in Lysyl Oxidase
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LysTyr
NH
CH CH2CH2CH2CH2
CO
OH
CH2
NH
O
O
CHCONHNH
Asp-Thr Asn-Ala-Asp
3.135
Val-Ala-Glu-Gly-His
Structure of Lysine Tyrosylquinone in Lysyl Oxidase
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Enzymes Containing Amino Acid Radicals
Scheme 3.82
Mechanism proposed for galactose oxidase using a covalently bonded cysteine cross-linked tyrosine radical
Tyr272
O
SCys228
Tyr
O
SCys
Tyr
O
SCys
Tyr
O
SCys
Tyr
OH
SCys
H R
O
Tyr
OH
SCys
H R
O
Tyr
OH
SCys
Tyr
O
SCys
H R
H OH
H R
H OH
H R
H O
H R
H O
H R
O
H R
O
.
.
ER2; radical E2concerted mechanism
..
.
3.136
H atom transferstepwise mechanism
Cu(II)+
H
ketylradical anion
Cu(II)++
Cu(II)++
Cu(II)++
Cu(II)++Cu(I)+ Cu(I)+
O2O2
-H+
-RCHO
-HOO-
Cu(II)++
O2
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Scheme 3.83
quadricyclane analogue
norbornadiene analogue
[,-2H2] 3.137 kH/kD = 6 on inactivation 1e- reduced form
Mechanism-based Inactivation of Galactose Oxidase by Hydroxymethylquadricyclane and
Hydroxymethylnorbornadiene
ketyl radicals
CH2O-HC
O
H C
O
H
C
O
H
CH2OH
Tyr272
OS
Cys228
same as with 3.137
Tyr
OHS
Cys
.
3.136
3.138
3.137
3.139
-B
CH2O
Tyr
OHS
Cys
inactivated enzymecomplex
Cu(II)++Cu(II)++
Cu(II)++
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OHN
Me
OCDP
OH
O
O
Me
OCDPOH
Pyr
OMe
OCDPOH
O OH
NH2
OH
OHN
Me
OCDPOHPyr
O
OCDPOH
OH
HO
HO
OHN
Me
OCDPOHPyr
N
OH=O3PO
+
O
O MeOCDP
OH
+
3.143
3.140 NADH, FADPyr = pyridine ring of PMP
O
MeOCDP
OH
+
3.142NADPH
HO
3.141
3.142
3.1443.1453.1463.147
-H2O
Fe(III)Fe(II)S2
NAD+
Scheme 3.84
Iron-sulfur Clusters and Pyridoxamine 5-Phosphate (PMP)Biosynthesis of ascarylose
E1
E1/E3*
ascarylose
Reaction catalyzed by CDP-6-deoxy-L-threo-D-glycero-4-hexulose-3-dehydratase (also called E1) and CDP-6-
deoxy-Δ3,4-glucoseen reductase (also called E3)
(PMP)
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Usually in carbanionic reactions of amino acids
With E1/E3 PMP may be involved in two one-electron reductions (EPR)
3.142
N
CH2NH2
OH
CH3
=O3PO
Pyridoxamine 5-Phosphate (PMP)
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[2Fe-2S] [3Fe-4S] [4Fe-4S]
1 electron and 2 electron transfers
3.1503.148 3.149
FeS
Fe
S
S
S
S
S S
Fe
S Fe
S
Fe
S
S
S
S
Cys
Cys
Cys
Cys
Cys
Cys
CysS
Fe
S Fe
S
Fe
S
S
S
S
Cys
Cys
Cys
FeS Cys
Iron-sulfur Clusters
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HN
O H
N
=O3PO
Me
Me
O
OH
OCDP
OCDPOH
O
N
Me
Me
=O3PO
HO
HN
HN
O- H
N
=O3PO
Me
MeO
OH
OCDPO
Me
O
OHOCDP
OH
OCDPOH
O
N
Me
Me
=O3PO
HO
HN
OCDPOH
O
Me
O
E3
3.151
E1, PMPE3, NADH E3
+
+
E1
E1
+ +
+ +
HN
O- H
N
=O3PO
Me
MeO
OH
OCDP
++
OH
H
B:
E3
BH
PMP
E1+
B
H
+
-PMP
3.145
H2O
NAD+
NADH
Fe(III)2S2Fe(III)Fe(II)S2
Fe(III)2S2
Fe(III)Fe(II)S2
H+
Fe(III)Fe(II)S2Fe(III)2S2
Fe(III)Fe(II)S2
Fe(III)2S2FADH
FADH-
FAD
Scheme 3.85
1e- transfer
*
**
* In 3H2O, 1 3H in product EPR evidence
1e- transfer
Mechanism Proposed for the Reduction of CDP-6-deoxy-Δ3,4-glucoseen by E1 and E3
** (4R)- and (4S)-[4-3H]NADH both transfer 3H 3H released as 3H2O
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Molybdoenzymes and Tungstoenzymes
HN
N NH
HN S
MoVIS
OPO3=
HO
O
H2N
O O
3.152HN
N NH
HN S
MoVIS
OO
O
H2N
S S
O
NH
HN
NH
NO
PO
PO
O
OH OH
N
N
N
HN
H2N
O
O
O-
O
O-
NH2
O
PO
PO
O O
O-O-O
N
OH OH
N
N
NH
O
NH2
3.153
3.154
HN
N NH
HN S
WVIS
OPO3=
O
O
H2N
S S
O
NH
HN
NH
NO3
=PONH2
O
Hydroxylation generally by flavin, heme, pterin enzymes (next chapter)with the O coming from O2; in these enzymes, the O comes from H2O
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Scheme 3.86
Mechanism for Sulfite Oxidase (in liver)
HN
N NH
HN S
MoVIS
OPO3=
HO
O
H2N
O O
OS
O
O HN
N NH
HN S
MoVIS
OPO3=
HO
O
H2N
O
O
O
S O-
O
HN
N NH
HN S
MoIVS
OPO3=
HO
O
H2N
O
O
O
SO-O
H OHB:
HN
N NH
HN S
MoIVS
OPO3=
HO
O
H2N
O
O
O
SO-
O
OH
HN
N NH
HN S
MoIVS
OPO3=
HO
O
H2N
:
O
3.152
O
-2e- -SO4=
O from H2O
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Scheme 3.89
HydrogenasesThe only known non metallohydrogenase
pro-R specific
Reduction with No Cofactors
14a
H2N
HN
N
N
N
N
CH3
CH3
H
O
H
H2N
HN
N
N
HN
N
CH3
CH3
H
O
HR HS
+ H2
3.158 3.159R R
+
+
H
H+
Reduction of N5,N10-methenyl tetrahydromethanopterin to N5,N10-methylene tetrahydromethanopterin catalyzed by the
hydrogenase from a methanogenic archaebacterium
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Scheme 3.91
Model Study for Metal-free Hydrogenase
110 °C
strong acid
irreversibleantiperiplanar stereoelectronic effect
Reaction of perhydro-3a,6a,9a-triazaphenalene with tetrafluoroboric acid
NN N
NN N
+ H++ H2
3.161
+
3.162H
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Scheme 3.90
initially, not resonance stabilized
conformational change
Mechanism Proposed for Oxidation of N5,N10-methylene tetrahydromethanopterin to
N5,N10-methenyl tetrahydromethanopterin (reverse of the reaction in Scheme 3.89)
OO
H
O-O
H H
NN
RHH3C
ringH
HS
ring N
N
ring
H
H3CH
ring
HR
NN
RHH3C
ringH
H
ringH
R
3.159 3.160
+
3.158
++ H2