Title: The%20Organic%20Chemistry%20of%20Enzyme%20Catalyzed%20Reactions%20%20%20Chapter%203
1The Organic Chemistry of Enzyme-Catalyzed
Reactions Chapter 3 Reduction and Oxidation
2Redox Without a Coenzyme
3Reaction Catalyzed by Glyoxalase
Scheme 3.1
methylglyoxal
lactic acid
Looks like a Cannizzaro reaction
4Cannizzaro Reaction Mechanism
Scheme 3.2
5Reactions Catalyzed by Glyoxalase I and
Glyoxalase II
reduced
oxidized
glutathione
Scheme 3.3
6Glutathione (GSH)
7Hydride Mechanism for Glyoxalase
oxidized
reduced
Scheme 3.4
Intramolecular Cannizzaro reaction
8- 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
9Enediol Mechanism for Glyoxalase
cis-enediol
Scheme 3.5
10Reaction of Glyoxalase with Fluoromethylglyoxal
Another test for the mechanism
Scheme 3.6
same oxidation state
11Hydride Mechanism for the Reaction of Glyoxalase
with Fluoromethylglyoxal
Scheme 3.7
12Enediol Mechanism for the Reaction of Glyoxalase
with Fluoromethylglyoxal
Scheme 3.8
13Hydride Mechanism for the Reaction of Glyoxalase
with Deuterated Fluoromethylglyoxal
Scheme 3.9
F- loss decreased
deuterium isotope effect
14Enediol Mechanism for the Reaction of Glyoxalase
with Deuterated Fluoromethylglyoxal
deuterium isotope effect
Scheme 3.10
F- loss increased
15increased F- loss supports enediol mechanism
16Redox Reactions that Require CoenzymesNicotinami
de 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)
17NAD(P)
NAD(P)H
Enzyme without coenzyme bound - apoenzyme Enzyme
with coenzyme bound - holoenzyme
coenzyme
apoenzyme holoenzyme
Called reconstitution
18Abbreviated Forms
NAD(P)H (reduced)
NAD(P) (oxidized)
19- 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)
20Biosynthesis of Nicotinamide Adenine Dinucleotide
(NAD)
nicotinic acid (vitamin B3) niacin
from ATP
Scheme 3.11
21Reactions Catalyzed by Pyridine
Nucleotide-containing Enzymes
Oxidation potential NAD/NADH is -0.32 V
Figure 3.1
22Reactions Catalyzed by Alcohol Dehydrogenases
Mechanism
Scheme 3.12
In 3H2O, no 3H in NAD(P)H
Hydride mechanism
23Reaction Catalyzed by Alcohol Dehydrogenases
Using Labeled Alcohol
Scheme 3.13
No H found in H2O
Supports hydride mechanism
24Cyclopropylcarbinyl Radical Rearrangement
Test for a radical intermediate
Scheme 3.14
25Test for the Formation of a Radical Intermediate
with Lactate Dehydrogenase
Scheme 3.15
No ring cleavage - evidence against radical
mechanism
26Chemical Model for the Potential Formation of a
Cyclopropylcarbinyl Radical during the Lactate
Dehydrogenase-catalyzed Reaction
Scheme 3.16
Should have seen ring opening in the enzyme
reaction if a cyclopropylcarbinyl radical formed
27Nonenzymatic Reduction of ?-Chloroacetophenone
Another test for a radical intermediate
Nonenzymatic reaction
radical reduction product
Scheme 3.18
28Horse Liver Alcohol Dehydrogenase-Catalyzed
Reduction of ?-Haloacetophenones
Scheme 3.19
hydride reduction product (stereospecific) X
F, Cl, Br
Supports no radical intermediate
When X I, get mixture of 3.25 (X I)
Electron transfer is possible if the reduction
potential is low enough
(radical reduction product)
29Stereochemistry
An atom is prochiral if by changing one of its
substituents, it changes from achiral to chiral
30Stereochemistry Determination of the chirality
of an isomer of alanine
R,S Nomenclature
Figure 3.2
31Determination of Prochirality
Figure 3.3
32Determination 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
33Determination of Carbonyl and Alkene (sp2)
Chirality
Figure 3.4
34Reaction of Yeast Alcohol Dehydrogenase (YADH)
with (A) 1,1-2H2ethanol and NAD and (B)
Ethanol and 4-2HNAD
Scheme 3.20
35Reaction of YADH with (A) 4-2HNAD2H Prepared in
Scheme 3.20A (B) Reaction of YADH with
4-2HNAD2H Prepared in Scheme 3.20B (C)
Reaction of YADH with 3.28 and NAD
No 2H
stereospecific
No H
Scheme 3.21
36only one H is transferred
re-face
37(A) Reaction of YADH with 1,1-2H2ethanol and
NAD (B) Reaction of glyceraldehyde-3-phosphate
dehydrogenase (G3PDH) with the cofactor produced
in A and glycerate 1,3-diphosphate
Not all enzymes transfer the same hydride
pro-R
pro-S transferred
Scheme 3.22
38Anti- and syn- conformations of NADH
Transition State for Hydride Transfer
Figure 3.5
syn-axial electrons assist
Boat-like TS
39Boat-boat equilibria of NADH
The enzyme may drive equilibrium
Figure 3.6
40Possible mechanism for the reaction catalyzed by
glutamate dehydrogenase
Oxidation of Amino Acids to Keto Acids
Hydride transfer
Scheme 3.24
41(A) Covalent catalytic mechanism for the
oxidation of aldehydes by aldehyde
dehydrogenases (B) noncovalent catalytic
mechanism for the oxidation of aldehydes by
aldehyde dehydrogenases
Oxidation of Aldehydes to Carboxylic Acids
covalent catalysis
Hydride transfers
Scheme 3.25
via hydrate
42Mechanism for the oxidation of inosine
5?-monophosphate by inosine 5?-monophosphate
dehydrogenase
Oxidation of Deoxypurines to Purines
inosine MP
Scheme 3.27
xanthine MP
43An Atypical Use of NADReaction catalyzed by
urocanaseNAD in a Nonredox Reaction
Scheme 3.28
44Urocanase Reaction Run with a 13C
Pseudo-substrate
apo-urocanase reconstituted with 13CNAD
substrate
exchangeable proton
45Adduct Isolated after Chemical Oxidation
NMR determined
46Mechanism Proposed for Urocanase
solvent incorporated
exchangeable
Scheme 3.29
47Biosynthetic conversion of riboflavin to FMN and
FAD
Flavin Coenzymes
riboflavin (vitamin B2)
FMN
FAD
Scheme 3.31
48Interconversion of the Three Oxidation States of
Flavins
oxidized
semiquinone
reduced
some covalently attached to The protein at these
positions
Scheme 3.32
49Redox Reactions Catalyzed by Flavin-dependent
Enzymes
Figure 3.8
50Oxidases vs. Dehydrogenases
Mechanisms for an oxidase-catalyzed oxidation of
reduced flavin to oxidized flavin
Scheme 3.33
only if spin inversion occurs
Oxidases use O2 for reoxidation of reduced flavin
coenzyme
51Mechanism for a dehydrogenase-catalyzed oxidation
of reduced flavin to oxidized flavin
Dehydrogenases Use Electron Transfer Proteins to
Reoxidize Reduced Flavin
Scheme 3.34
52Mechanisms for Flavoenzymes
Overall reaction of flavoenzymes
Scheme 3.35
53Mechanisms 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
54Two-Electon Mechanism (Carbanion)
D-Amino acid oxidase (DAAO) catalyzes the
oxidation of D-amino acids to ?-keto acids and
ammonia
55Evidence for MechanismIonization of substituted
benzoic acidsHammett Study
Derivation of the Hammett Equation
Scheme 3.36
As X becomes electron withdrawing, equilibrium
constant (Ka) should increase
56Reaction of hydroxide ion with ethyl-substituted
benzoates
A Similar Relationship Should Exist for a Rate
Constant (k) where Charge Develops in the
Transition State
Scheme 3.37
As X becomes electron withdrawing, rate constant
(k) should increase
57If 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
58Linear Free Energy RelationshipExample of a
Hammett plot
Figure 3.9
Ortho-substituent points are badly scattered
because of steric interactions and polar effects
59Hammett Relationship (Equation)
log k/k0 ?log K/K0 (3.3) log k/k0
?? (3.4)
reaction constant
electronic parameter (substituent constant)
EWG EDG -
- slope carbocation mechanism slope
carbanion mechanism
depends on type of reaction and reaction
conditions
depends on electronic properties of X ?H 0
60Application of Hammett Equation to Study of an
Enzyme Mechanism
D-Amino acid oxidase
? 5.44
? 0.73
Effect of X diminished by -CH2-
X EWG, Vmax
carbanionic TS
61Proposed Intermediate in the D-amino Acid
Oxidase-catalyzed Oxidation of Substituted
Phenylglycines
Scheme 3.38
What is the function of the flavin?
62Further Evidence for a Carbanion Intermediate
DAAO-catalyzed oxidation of ?-chloroalanine under
oxygen and under nitrogen
Scheme 3.39
expected elimination product
exclusive (in N2)
exclusive (in O2)
40 60 (in air)
Total amount of product(s) is the same under all
conditions
63Where on the flavin does the nucleophilic attack
occur?
Evidence against C4a addition
Nonenzymatic reaction of benzylamine with
N5-ethylflavin
Scheme 3.40
No adduct detected enzymatically
64Evidence for N5 AdditionReverse reaction
catalyzed by AMP-sulfate reductase
detected in absence of AMP
Scheme 3.41
65Initial Evidence for N5 Attack and for
Two-electron Chemistry
NADH-dependent reduction of 5-deazaflavin by
various flavoenzymes
5-deazaflavin
Scheme 3.42
66Comparison of Reduced 5-Deazaflavin with Reduced
Nicotinamide
Inappropriate flavin substitute
Favors 2-electron reactions because of
resemblance to NADH
Figure 3.10
67Support for Covalent Carbanionic Mechanism with
DAAO rather than Electron Transfer Mechanism
Inverse 2 deuterium isotope effect therefore
sp2 sp3 in TS, consistent with conversion
to carbanion and nucleophilic addition
68Covalent Carbanion versus Radical Mechanisms for
DAAO (Hammett study suggested carbanionic)
favored
Scheme 3.43
No base in crystal structure, but ?-H in line
with flavin Not clear how proton is removed
69Carbanion Mechanism Followed by 2 One-electron
Transfers
Reaction catalyzed by general acyl-CoA
dehydrogenase
Scheme 3.46
70Initial Mechanism Proposed for Mechanism-based
Inactivation of General Acyl-CoA Dehydrogenase by
(Methylenecyclopropyl)acetyl-CoA
Scheme 3.47
Mechanism-based inactivator
71Electron transfer mechanism for inactivation of
general acyl-CoA dehydrogenase by
(methylenecyclopropyl)acetyl-CoA
Evidence for Radical Intermediates
only pro-R removed
consistent with a radical pathway
Both enantiomers inactivate
Scheme 3.48
72Mechanism proposed for formation of 3.73 during
oxidation of (methylenecyclopropyl)acetyl-CoA by
general acyl-CoA dehydrogenase
Other Evidence for Radical Intermediate
isolated
Scheme 3.49
73Carbanion Followed by Single Electron Mechanism
for General Acyl-CoA Dehydrogenase
Not in text
74Possible mechanisms for monoamine
oxidase-catalyzed oxidation of amines
Single Electron Transfer Mechanism
-
Scheme 3.50
either Fl or amino acid residue
75Mechanism Proposed for Generation of an
Active-site Amino Acid Radical during Monoamine
Oxidase-catalyzed Oxidation of Amines
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.
76Cyclopropylaminyl Radical Rearrangement
Scheme 3.52
77Evidence for Aminyl Radical (radical
cation?)Mechanisms proposed for inactivation of
MAO by 1-phenylcyclopropylamine
All products derived from cyclopropyl ring opening
Scheme 3.53
78Chemical Reactions to Characterize the Structure
of the Flavin Adduct Formed on Inactivation of
MAO by 1-Phenylcyclopropylamine
Baeyer-Villiger reaction
Scheme 3.54
79Inactivation of MAO and Peptide Mapping
Cys-365
MALDI-TOF gives mass corresponding to X as
80Mechanism Proposed for Inactivation of MAO by
N-cyclopropyl-?-methylbenzylamine
Scheme 3.55 (modified)
81Mechanism proposed for MAO-catalyzed oxidation of
1-phenylcyclobutylamine and inactivation of the
enzyme
Further Evidence for Aminyl Radical (radical
cation?) Intermediate
Scheme 3.56
82Oxidation of (aminomethyl)cubane by MAO
Evidence for ?-Carbon Radical Intermediate
Gives product of a cubylcarbinyl radical
intermediate
detected
Scheme 3.57
83Reactions to Differentiate a Radical from a
Carbanion Intermediate
Scheme 3.58
84Mechanism proposed for MAO-catalyzed oxidation of
cinnamylamine-2,3-epoxide
Further Evidence for ?-Carbon Radical with MAO
isolated
Scheme 3.59
No products of a two-electron epoxide ring
opening detected
85Mechanism proposed for MAO-catalyzed
decarboxylation of cis- and trans-5-(aminomethyl)-
3-(4-methoxyphenyl)-2-14Cdihydrofuran-2(3H)-one
More Evidence for ?-Carbon Radical
isolated
detected
Scheme 3.60
evidence for reversible e- transfer (Fl ? Fl ,
Fl ? Fl)
-
-
86Mechanism proposed for inactivation of MAO by
(R)- or (S)-3-3Haryl-5-(methylaminomethyl)-2-oxa
zolidinone
Evidence for a Covalent Intermediate
Scheme 3.61
When x 3 and y 14, both radiolabels are
incorporated into the protein
87Reaction catalyzed by UDP-N-acetylenolpyruvylgluco
samine reductase (MurB)
Example of a Hydride Mechanism
2nd step in bacterial peptidoglycan biosynthesis
UDP-N-acetylmuramic acid
EP-UDP-GlcNAc
Scheme 3.63
88Hydride Mechanism for a Flavoenzyme (MurB)
In situ generation of FADH
Scheme 3.64
89MurB-catalyzed reduction of (E)-enolbutyryl-UDP-Gl
cNAc with NADP2H in 2H2O
Evidence for the Hydride Mechanism
anti-addition
extra Me for stereochemical determination
Scheme 3.65
A radical mechanism is not expected to be
stereospecific
90Conversion to 2-hydroxybutyrate of the product
formed from MurB-catalyzed reduction of
(E)-enolbutyryl-UDP-GlcNAc with NADP2H in 2H2O
Determination of the Stereochemistry of 3.108
D-configuration
Scheme 3.66
Substrate for D-lactate dehydrogenase but not
L-lactate dehydrogenase, therefore 2R
stereochemistry
91Enzymatic Syntheses of (2R,3R)- and
(2R,3S)-isomers of 2,3-2H2hydrobutyrate for NMR
Comparison with 3.109
omit ATP
Scheme 3.67
92Stereochemistry of the MurB-catalyzed Reduction
of (E)-enolbutyryl-UDP-GlcNAc
re-face
Scheme 3.68
93Reaction Catalyzed by Dihydroorotate Dehydrogenase
Scheme 3.69
D isotope effects on both Hs therefore concerted
94Unusual Reaction Catalyzed by a Flavoenzyme
UDP-galactopyranose mutase (UGM)
Requires FAD only reduced enzyme is active
When UGM was incubated with UDP-3H-galactopyrano
se 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
95UDP-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.
96Mechanism of UDP-galactopyranose mutase (UGM)
Mansoorabadi, S. O. Thibodeaux C. J. Liu, H.-w.
J. Org. Chem.. 2007, 72, 6329-6342.
97Synthesis of flavopapain
Artificial Enzyme (Synzyme)
catalyzes oxidation of NADH to NAD
papain
Scheme 3.70
98Unusual Reaction Catalyzed by Urate Oxidase
No flavin, but substrate reacts like a flavin
compare structures
detected
comes from H2O, not O2 (using 18O)
Scheme 3.71
reduced flavin
99Mechanism for an Oxidase-catalyzed Oxidation of
Reduced Flavin to Oxidized Flavin for Comparison
with Urate Oxidase
Scheme 3.33
100Possible Mechanism for the Urate
Oxidase-catalyzed Oxidation of Urate
detected
Scheme 3.72
Just like mechanism for oxidation of reduced
flavin by O2
101Pyrroloquinoline Quinone Coenzymes (PQQ)
Bound to quinoproteins
Also called methoxatin, coenzyme PQQ
102Possible Mechanisms for the Glucose
Dehydrogenase-catalyzed Oxidation of Glucose
Nucleophilic mechanism
from model study with MeOH C-5 favored over C-4
addition
Hydride mechanism
Scheme 3.73
From crystal structure, hydrogen over C-5
carbonyl, suggesting hydride mechanism
103Schiff base mechanism proposed -- NaCNBH3
inactivates the enzyme in the presence of
substrate
Evidence for Nucleophilic Mechanism for Plasma
Amine Oxidase
Plasma amine oxidase (contains CuII)
originally thought it was a PQQ enzyme (We will
see it is not)
3H isotope effect
1 equiv. 14C no 3H from NaCNB3H3
Scheme 3.74
Therefore excludes oxidation to 14PhCHO followed
by Schiff base formation with a Lys
104Stereochemistry of the reaction catalyzed by
plasma amine oxidase (PAO)
Isotope Labeling Shows Syn Hydrogens are Removed
(one-base mechanism)
PQQ is not the actual cofactor for PAO
Scheme 3.75
105Topa Quinone (TPQ), 6-Hydroxydopa, is the Actual
Cofactor for PAO
Characterized by Edman degradation, and mass,
UV-vis, resonance Raman, and NMR spectrometries
106Plasma amine oxidase-catalyzed amine oxidation
with topa quinone shown as the cofactor
Using a Hammett study showed ? 1.47 0.27
(carbanion-like TS)
Scheme 3.76
107Model Study for Topa Quinone
C-5
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
108Chemical Model Study for the Mechanism of Topa
Quinone-dependent Enzymes
Scheme 3.77
Deactivates C-2 and C-4 carbonyls, so C-5
carbonyl is more reactive
109Detailed Mechanism Proposed for Topa
Quinone-dependent Enzymes
Mechanism for Plasma Amine Oxidase
Scheme 3.79
110Mechanism Proposed for Reoxidation of Reduced
Topa Quinone
Based on EPR spectroscopy
detected
Scheme 3.80
111Mechanism Proposed for Biosynthesis of Topa
Quinone from Tyrosine
Scheme 3.81
Topa quinone is ubiquitous - found in bacteria,
yeast, plants, mammals
112Tryptophan Tryptophylquinone Coenzyme
Observed by X-ray analysis
in methylamine dehydrogenase Hammett study with
?
(carbanion mechanism)
113Coenzyme in Lysyl Oxidase
Isolated from a proteolytic digestion
114Structure of Lysine Tyrosylquinone in Lysyl
Oxidase
Lys
Tyr
115Mechanism proposed for galactose oxidase using a
covalently bonded cysteine cross-linked tyrosine
radical
Enzymes Containing Amino Acid Radicals
Scheme 3.82
116Mechanism-based Inactivation of Galactose Oxidase
by Hydroxymethylquadricyclane and
Hydroxymethylnorbornadiene
Scheme 3.83
quadricyclane analogue
ketyl radicals
norbornadiene analogue
?,?-2H2 3.137 kH/kD 6 on inactivation
1e- reduced form
117Reaction catalyzed by CDP-6-deoxy-L-threo-D-glycer
o-4-hexulose-3-dehydratase (also called E1) and
CDP-6-deoxy-?3,4-glucoseen reductase (also called
E3)
Iron-sulfur Clusters and Pyridoxamine
5?-Phosphate (PMP) Biosynthesis of ascarylose
(PMP)
E1
E1/E3
ascarylose
Scheme 3.84
118Pyridoxamine 5?-Phosphate (PMP)
Usually in carbanionic reactions of amino
acids With E1/E3 PMP may be involved in two
one-electron reductions (EPR)
119Iron-sulfur Clusters
3Fe-4S
4Fe-4S
2Fe-2S
1 electron and 2 electron transfers
120Mechanism Proposed for the Reduction of
CDP-6-deoxy-?3,4-glucoseen by E1 and E3
1e- transfer
1e- transfer
In 3H2O, 1 3H in product
EPR evidence
(4R)- and (4S)-4-3HNADH both transfer 3H
3H released as 3H2O
Scheme 3.85
121Molybdoenzymes and Tungstoenzymes
Hydroxylation generally by flavin, heme, pterin
enzymes (next chapter) with the O coming from O2
in these enzymes, the O comes from H2O
122Mechanism for Sulfite Oxidase (in liver)
O from H2O
Scheme 3.86
123Reduction with No Cofactors
Hydrogenases The only known non metallohydrogenase
Reduction of N5,N10-methenyl tetrahydromethanopter
in to N5,N10-methylene tetrahydromethanopterin
catalyzed by the hydrogenase from a methanogenic
archaebacterium
pro-R specific
Scheme 3.89
124Reaction of perhydro-3a,6a,9a-triazaphenalene
with tetrafluoroboric acid
Model Study for Metal-free Hydrogenase
110 C
strong acid
irreversible
antiperiplanar stereoelectronic effect
Scheme 3.91
125Mechanism Proposed for Oxidation of
N5,N10-methylene tetrahydromethanopterin to
N5,N10-methenyl tetrahydromethanopterin (reverse
of the reaction in Scheme 3.89)
initially, not resonance stabilized
conformational change
Scheme 3.90