the organic chemistry of enzyme-catalyzed reactions chapter 3 reduction and oxidation
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The Organic Chemistry of Enzyme-Catalyzed Reactions
Chapter 3
Reduction and Oxidation
Redox Without a Coenzyme
Internal redox reaction
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
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
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
Glutathione (GSH)
H3N CHCH2CH2
COO-
CNH
O
CH
CH2SH
C NHCH2CO2-
O(γ-Glu-Cys-Gly)
3.3
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
• 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
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
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
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
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
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
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-
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
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
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
Abbreviated Forms
NAD(P)+
(oxidized)NAD(P)H(reduced)
R
N
NH2
O
3.12
R
N
NH2
OHH
3.13
• 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)
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+)
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
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
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
Scheme 3.14
3.19
k = 108 s-1
3.20
Cyclopropylcarbinyl Radical Rearrangement
Test for a radical intermediate
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
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
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
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
Stereochemistry
An atom is prochiral if by changing one of its substituents, it changes from achiral to chiral
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)
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
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
Figure 3.4
Determination of Carbonyl and Alkene (sp2) Chirality
CH3C
O
H CH3C
CH2
H
si face
re face
si face
re face
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+
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+
only one H is transferred
re-face
N
R
NH2
OHRHS
NR
HR
HS
H2N
O
3.29
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
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‡
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
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
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
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
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
“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
NMR determined
N
NH2
O
R
N
HN COO-
13
13
3.43
Adduct Isolated after Chemical Oxidation
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
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
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
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
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
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
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
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
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
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
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
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
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
= +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-
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?
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
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
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+
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
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
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
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
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
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
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
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+
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
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
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
Scheme 3.52
Cyclopropylaminyl Radical Rearrangement
NR NR
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
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
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
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+
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
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
Scheme 3.58
Reactions to Differentiate a Radical from a Carbanion Intermediate
O
OR
R
O
RO
R
A
B
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
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
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+
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
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
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
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
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
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
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
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
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)
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)
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
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
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
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
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
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
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)
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
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
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‡)
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
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+
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
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
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+
in methylamine dehydrogenase
Hammett study with +
3.133
NH
NH
O
O
ProteinProtein
Tryptophan Tryptophylquinone Coenzyme
Observed by X-ray analysis
NH2X
(carbanion mechanism)
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
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
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
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)++
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)
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)
[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
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
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
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
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
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
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
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