Transcript The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 9 Isomerizations Isomerizations Conversion of one molecule into another with the same formula • Hydrogen shifts to the same.

The Organic Chemistry of
Enzyme-Catalyzed Reactions
Chapter 9
Isomerizations
Isomerizations
Conversion of one molecule into another with the
same formula
• Hydrogen shifts to the same carbon: [1,1]-H shift
• Hydrogen shifts to the adjacent carbon: [1,2]-H shift
• Hydrogen shifts to two carbon atoms away: [1,3]-H
shift
[1,1]-Hydrogen Shift
Racemase with no cofactors
Glutamate racemase
Not PLP - no visible absorbance
Not pyruvoyl - acid hydrolysis gave no pyruvate
No M2+ - EDTA has no effect
No acyl intermediates - no 18O wash out of [C18O2H]Glu
Not oxidation/reduction - 2H is incorporated into C-2 from
2H O
2
Therefore deprotonation/reprotonation mechanism
[1,1]-Hydrogen Shift
Amino acid racemases
(A) One-base mechanism for racemization (epimerization),
(B) Two-base mechanism for racemization (epimerization)
R
R
NH3
H
+
NH3+
-OOC
COO-
A
-OOC
H
NH3
+
-OOC
COO-
a
H
B
B
R
R
H
Scheme 9.1
H
B
B
B
R
+H N
3
BH
a
B
R
+H N
3
NH3+
b
b
H
B
-OOC
BH
H
B
One base: substrate proton transferred to product
Two base: incorporated proton from solvent
With Glu racemase: solvent deuterium in product, not substrate (B)
also, primary kinetic isotope effect with [2-2H]Glu
An “Overshoot” Experiment with (R)-(-)-glutamate to Test for
a Two-base Mechanism for Glutamate Racemase
in D2O
100
60
20
0
E
ll
ip
ti
ci
ty
(m
il
li
de
gr
ee
)
-20
-60
-100
1000
Figure 9.1
2000
Time (sec)
3000
Another Test for a Two-Base Mechanism
Elimination of HCl from threo-3-chloroglutamic acid by
the C73A and C184A mutants for glutamate racemase
COO- H
Cl
Cl
R
R
S
S
S
-OOC
COO-
H
H
NH3+
NH3+
C184
COO- H
C73A
COO-
COO-
COO-
H
H
Cl
R
R
-OOC
NH3+
NH3+
+H
COO-
3N
D2O
D
COO-
D2 O
COO-
D
H
Scheme 9.2
COO-
H
S
S
H
S
C73
9.1
C184A
Cl
COO-
H
H
-OOC
O
9.2
COO-
O
9.2
-OOC
H
NH3+
Proposed Mechanism for Proline Racemase
S
S
H
H
H
N
HO
HO
H
H
N
S
H
SH
O
S
H
N
HO
H
O
S H
H
N
HOOC

O
‡
S
H
S
9.3
Scheme 9.3
Inactivation by ICH2COO- only after a reducing agent
(RSH or NaBH4)
is added
Reduces active site disulfide to dithiol
Transition State Analogue Inhibitor
Because substrates bind tightest at the transition
state of the reaction, a compound resembling the
TS‡ structure would be more tightly bound
-OOC
H
N
9.4
resembles 9.3
TS‡ analogue
inhibitor for
Pro racemase
Pyridoxal 5-Phosphate (PLP) Dependent
Racemases
Proposed mechanism for PLP-dependent alanine racemase
H
CH3
COOCOO-
C
+ NH
PLP
CH3
H
C
Keq ~ 1
+
3
L-Ala
NH3
D-Ala
PLP
PLP
:B
B
H
COO-
H
CH3
C
+
CH3
CH3
C
+
NH
OH
=O PO
3
COO-
COO-
H
+ NH
NH
OH
=O PO
3
C
OH
=O PO
3
:
+
Scheme 9.4
N
H
CH3
CH3
N
H
quinonoid intermediate
Usually, a one-base mechanism
+
N
H
CH3
PLP was a coenzyme for decarboxylases
(break C-COOH bond) and now for
racemases (break C-H bond)
How can PLP enzymes catalyze
selective bond cleavage?
Stereochemical Relationship Between the -Bonds
Attached to C and the p-Orbitals of the -System
in a PLP-Amino Acid Schiff Base
H
R
-OOC
NH
NH
Figure 9.2
PLP
all sp2 + p atoms
The -bond that is parallel to (overlapping with)
the p-orbitals will break (C-H in this case)
Dunathan Hypothesis for PLP Activation of the Bonds
Attached to C in a PLP-Amino Acid Schiff Base
The rectangles represent the plane of the pyridine ring of the PLP.
The angle of viewing is that shown by the eye in Figure 9.2.
pyridine ring
of PLP
COO- +
H
R
C
N CH
COO-
A
+
N CH
+
C
R
H
R
B
C
N CH
-OOC
H
C
Figure 9.3
The -charge stops free rotation, which results in
selective bond cleavage
Other Racemases
Reaction catalyzed by mandelate racemase
HO
Ph
H
H
CO2-
Ph
R-mandelate
OH
CO2-
S-mandelate
Scheme 9.5
No internal return in either direction
With (R)-mandelate no -H exchange with solvent
With (S)-mandelate there is exchange with solvent
A Two-base Mechanism for Mandelate Racemase that Accounts
for the Deuterium Solvent Exchange Results.
Lys-166 acts on the (S)-isomer, and His-297 acts on the (R)-isomer
OH
OH
:
166Lys
A
ND2
O
(S)
ND
166Lys
O-
Ph
297His
D
H
H
+
N
D
D
O-
Ph
OH
O-
Ph
O
Mg2=
solvent
exchange
O
Mg2+
ND
297His
N
:
N
H
B
HO
H
OH
HO
O-
Ph
OPh
O
(R)
Mg2+
Scheme 9.6
H
O-
Ph
O
Mg2+
no solvent
exchange
O
H297N mutant is capable of exchanging the
-H of the S-isomer, but not the R-isomer
H297N Mutant Capable of Elimination of HBr from
(S)-9.5, but not from the (R)-isomer
Elimination of HBr from (S)-p-(bromomethyl)mandelate,
catalyzed by the H297N mutant of mandelate racemase
NH2
Lys-166
H
OH
COO-
Br
9.5
-HBr
OH
O
COO-
COO9.6
Scheme 9.7
K166R mutant catalyzes elimination of HBr from
the (R)-isomer, but not from the (S)-isomer
Epimerases
Peptide epimerases
Mechanism 1
Elimination/addition (dehydration-hydration) mechanism for peptide
epimerization
-OH
OH
OH
O
AcNH-Gly-Leu
H
Phe-Ala-OH
N
H
B
O
H
Phe-Ala-OH
N
H
AcNH-Gly-Leu
B
H
O
:B
O
H
O
Phe-Ala-OH
N
H
AcNH-Gly-Leu
O
9.7
:B
H
B
H
B
Scheme 9.8
H
O
AcNH-Gly-Leu
OH
OH
H
O
Phe-Ala-OH
N
H
AcNH-Gly-Leu
Phe-Ala-OH
N
H
O
B
O
H
With
18O
18O
in the Ser OH group, no loss of
as H2
Therefore, mechanism 1 is unlikely.
18O
:B
-Cleavage Mechanism for Peptide
Epimerization
Mechanism 2
O
H
O
O
AcNH-Gly-Leu
:B
H
O
B
N
H
AcNH-Gly-Leu
H
O
H
H
Phe-Ala-OH
H
Phe-Ala-OH
N
H
AcNH-Gly-Leu
9.8
H
Phe-Ala-OH
N
H
O
O
OH
O
H
:B
B
Scheme 9.9
10 mM NH2OH has no effect on product formation
Therefore, mechanism 2 is unlikely.
Deprotonation/Reprotonation Mechanism
Mechanism 3
B
B
O
AcNH-Gly-Leu
H
OH
B
O
H
H
H
Phe-Ala-OH
N
H
AcNH-Gly-Leu
B:
OH
Phe-Ala-OH
N
H
AcNH-Gly-Leu
H
H
B
H
H
O
O
O
HO
B
Phe-Ala-OH
N
H
O
:B
B
Scheme 9.10
In D2O D is incorporated into product, not substrate (short incubation;
monitored by electrospray ionization mass spectrometry)
Deuterium isotope effect for [-2H]-peptides in the L- to D-direction
is different from that in the D- to L-direction (two-base mechanism)
These results are consistent with mechanism 3.
Epimerization with Redox Catalysis
Proposed mechanism for dTDP-L-rhamnose synthase-catalyzed
conversion of dTDP-4-keto-6-deoxy-D-glucose (9.9) to dTDP-L-rhamnose
(9.10)
H
CH3
B+
H
O
O H
OHH
NADPH
HO
H
H
OdTDP
H
two different
enzymes
B+
O
H
B+ H
CH3
O H
H
H
OdTDP
OH
reductase
:B
CH3
H
OH
9.10
9.9
epimerase
B:
Scheme 9.11
OdTDP
OH
OH
B:
O H
H CH3 H
OH
B+
O
H
H
H
OH
H3C
O H
H
OdTDP
OH
H
B+
H O
H
O H
H
B:
OdTDP
OH
H
O
H
H
O H
H CH3 H
OdTDP
OH
O OH
OH
NH2
:
N
R
C-H cleavage at C-3 and C-5 show kinetic isotope effects (3.4 and 2.0,
respectively)
In 2H2O 2H incorporation at both C-3 and C-5
Partial exchange gives only C-3 proton exchange, never only C-5
proton exchange (ordered sequential mechanism)
UDP-Glucose 4-Epimerase
OH
O
OH
OH
O UDP
OH
9.11
UDP-glucose
OH
HO
O
OH
O UDP
OH
9.12
UDP-galactose
In H218O, no incorporation of 18O into product
No change in oxidation state, but is
deprotonation/reprotonation reasonable?
Tritium is incorporated from NAD3H into a derivative of the
suspected intermediate of the UDP-glucose 4-epimerasecatalyzed reaction
The enzyme requires NAD+; no exchange with solvent
without OH
CH3
reverse
reaction
CH3
O
E•NAD3H
+
O
HO
OH
O
OH
O dTDP
OH
3H
O dTDP
OH
9.13
proposed intermediate
Scheme 9.12
Proposed Mechanism for Reaction
Catalyzed by UDP-Glucose 4-Epimerase
NAD+
H
OH
O
O
O
OH
O UDP
OH
O
H
OH
OH
B H
HO
OH
OH
O UDP
OH
NAD H
9.14
O
H
NAD+
O UDP
OH
:B
Scheme 9.13
Evidence for 9.14: incubate enzyme with UDPgalactose,quench with NaB3H4.
3H at C-4 of both UDP-glucose
and UDP-galactose
Mechanism to Account for Transfer of Hydrogen
from the Top Face of UDP-glucose and Delivery
to the Bottom Face of the 4-Keto Intermediate
OH
H2N
OH
O
O H
R N
OH
O
H
H2N
O
UDP
O
H
H
O
B
OH
O
R N
H
Scheme 9.14
OH
H
HO
HO
HO
:B
-NAD+
O
UDP
OH
O
HO
O
UDP
Mechanistic Pathway for the GDP-D-mannose-3,5epimerase-catalyzed Conversion of GDP-D-mannose
(9.15) to GDP-L-galactose (9.18)
No change in oxidation state, but NAD+ required
OH
H
OH
O
OH
NADH
O
O
OH OH
O
O GDP
OH
9.16
9.15
H
NAD+
O
OHOH
OH
Scheme 9.15
O
OH
O GDP
O GDP
OH
OH
OH
NAD+
OH
9.18
NADH
O
O
OH OH
O GDP
O GDP
OH
9.17
[1,2]-H Shift
Reaction catalyzed by aldose-ketose isomerases
Lobry de Brun-Alberda von Ekenstein Reaction
H
CHO
CH2OH
C
C
R
9.19
Scheme 9.16
OH
R
9.20
O
Two Mechanisms
Mechanism 1
cis-Enediol mechanism for aldose-ketose isomerases
cis-enediol
H
H
B:
H*
C
O
H
B
B
H*
OH
R
R
re OH
C
C
re
O
:
C
:B
C
H
(2R)
B
OH
O
H
R
(2R)
Scheme 9.17
B
H*
H
R
OH
O
:B
O
H
B
H
B
9.22
H*
R
OH
R
O
:
H*
H
OH
R
suprafacial
transfer of H
9.21
B:
B
H
pro-R
*
H C
B
H
H
Mechanism 2
Hydride transfer mechanism for aldose-ketose isomerases
H
H
C
O
H* C
O
H
H
R
B
:B
*H
H
C
O
C
O
R
H
:B
B
Scheme 9.18
Partial incorporation of solvent observed inconsistent with hydride mechanism
[1,3]-H Shift
Enolization
Reaction catalyzed by phenylpyruvate tautomerase
R
R
O
OH
CO2HS HR
R = H or OH
Scheme 9.19
CO2HS
removes pro-R hydrogen
Two Conformers Possible
Conformations of phenylpyruvate that would form
Z- and E-enols by phenylpyruvate tautomerase
O
H
B
OH
anti
CO2-
CO2Z
HS HR
B:
CO2-
CO2-
syn
O
HS HR
H
B
B:
Scheme 9.20
OH
E
To Test for Favored Conformation
R
R
CO2-
F
R
9.24
CO2-
CO2-
F
CO29.23
R
9.25
9.26
favored inhibitors
Therefore syn geometry to
E enol most likely
Allylic Isomerizations
Mechanism 1
Carbanion mechanism for allylic isomerases
H
B:
Scheme 9.21
B
H
H
This H could come
from the substrate (if
no solvent exchange)
Mechanism 2
Carbocation mechanism for allylic isomerases
H
H
H
B+
Scheme 9.22
H
H
B:
This H comes from
solvent, not from the
substrate
Mechanism 3
[1,3]-Sigmatropic hydride shift mechanism
for allylic isomerases
H
H
Scheme 9.23
Unlikely -- [1,3]-hydride shift is allowed antarafacial,
which is geometrically impossible
Carbanion Mechanism
Reaction catalyzed by 3-oxo-5-steroid isomerase
Scheme 9.24
O
O
1
2
3
O
4
H
(D)
5
6
H H
9.27
O
H
H
(D)
H
9.28
Principal reaction transfers 4-H to
6-position; therefore suprafacial
Eliminates carbocation mechanism and [1,3] hydride shift
Evidence for an Enol Intermediate in the Reaction
Catalyzed by 3-Oxo-5-steroid Isomerase
O
O
enzymatic
O
O
9.32
9.31
O
O
non
enzymatic
pH 4.5
HO
9.33
enzymatic
9.32
Scheme 9.26
9.31
(90%)
+
O
9.34
10%
Kinetic Competence of Enol
Further evidence for an enol intermediate in the
reaction catalyzed by 3-oxo-5-steroid isomerase
O
HO
9.35
O
O
O
O
9.36
Scheme 9.27
same rates
9.37
From Site-directed Mutagenesis, Tyr-14 is
the Acid and Asp-38 the Base
Asp-38
O
O
H
H O
Tyr-14
suprafacial
H
O
H O
H O
Tyr-14
Tyr-14
orthogonal (favored)
antarafacial
9.38
from NOE studies
Reactions Designed to Investigate the Function of Tyr-14
at the Active Site of 3-oxo-5-steroid Isomerase
To probe the function of Tyr-14
OH
Uv spectrum bound
to enzyme is same
as neutral amine.
OH
+ H+
+
H2 N
Therefore Tyr-14 does
not protonate C-3
carbonyl
H3N
9.39
O
O
- H+
-O
HO
9.40
Scheme 9.28
Structure bound to
enzyme even at low
pH (pKa of the
phenol must be very
low).
Therefore Tyr-14
H bonds to dienolate
Carbanion Mechanism
Mechanism for suprafacial transfer of the 4-proton to
the 6-proton of steroids catalyzed by 3-oxo-5-steroid
isomerase
O
H
_
O
O
O
O
O
2H
H
H H
COO-
O
Scheme 9.29
O
COO 2H
Tyr-14
Tyr-14
O
2H
H
COOAsp-38
Asp-38
Asp-38
Tyr-14
H
Asp-99 Located Adjacent to Tyr-14
One mechanism for the function of Asp-99 in
the active site of 3-oxo-5-steroid isomerase
O
Tyr14O
H
O
Tyr14O
Tyr14O
H
O
Asp99 COO
O
H
O
H
O
H
H
O
38Asp
Scheme 9.30
Asp99 COO
H
O
H
O
O
38Asp
H
H
H
Asp99 COO
O
H
O
38Asp
H
Crystal structure with equilenin bound is consistent with
Asp-99 and Tyr-14 both coordinated to oxyanion
O
HO
9.41
equilenin
4-Oxalocrotonate Tautomerase
CO2-
CO2-
O
CO29.42
CO2-
OH
CO29.43
O
CO29.44
Scheme 9.32
From deuterated substrates, substrate analogues,
and reactions run in D2O, 9.42 to 9.44 is suprafacial
(one-base mechanism)
Carbocation Mechanism
Reaction catalyzed by isopentenyl
diphosphate isomerase
O
O
O P O P OOO9.45
isopentenyl diphosphate
O
Mg++
O
O P O P OOO9.46
dimethylallyl diphosphate
Scheme 9.33
No exchange of solvent into substrate, only into product
One base mechanism
Evidence for a Carbocation Mechanism
OP2O63-
OP2O63-
CF3
HN+
9.49
9.48
rate is 1.8  10-6 times
OP2O63-
Ki = 14 pM
transition state
analogue inhibitor
Proposed Mechanism for Isopentenyl
Diphosphate Isomerase
OPP
B
2H
OPP
2H
OPP
H
2H
B:
Scheme 9.35
Aza-allylic Isomerization
H
+NH
+NH
H
Scheme 9.36
PLP-dependent Aminotransferase
Reaction catalyzed by L-aspartate aminotransferase
O
H
-OOC
CH2
14C
COO-
+
15 NH +
3
Scheme 9.37
CH3 13C
COO-
H218O
H
-OOCCH 14C
2
18O
COO-
+
CH3
13C
COO-
15NH +
3
First Half Reaction Catalyzed by Aspartate
Aminotransferase
B:
H
NH
OH
=O PO
3
-OOC
H
-OOC
N
H
14C
COO-
14C
see
Scheme 8.39
15NH
15NH
2
slow step
OH
=O PO
3
9.50
COO-
N
H
aldimine
-OOC
B H
14C
COO-
18O
-OOC
14C
15NH
COO-
15NH
9.51
H218O
15NH
3
OH
=O PO
3
..
N
H
quinonoid
Scheme 9.38
COO-
-OOCCH 14C
2
=O
OH
3PO
OH
=O PO
3
N
H
N
H
9.52
PMP
Second Half Reaction Catalyzed
by Aspartate Aminotransferase
H
B
15NH
3
CH3
O
OH
=O PO
3
CH3
N
H
13C
B:
COO-
OH
=O PO
3
N
H
H
H
CH3
13C
COO-
CH3
OH
=O PO
3
N
H
13C
COO-
15NH
15NH
15NH
H
9.53
9.52
13C
COO-
CH3
COO-
13C
15NH
3
NH2
OH
=O PO
3
NH
N
H
OH
=O PO
3
N
H
Scheme 9.39
This is the reverse of the mechanism in Scheme 9.38
Crystal structures of:
• native enzyme with PLP bound
• substrate reduced onto PLP
• enzyme with PMP bound
All are consistent with mechanisms
in Schemes 8.39 and 9.38
Evidence for Quinonoid Intermediate
OH
COO-
-OOC
+ NH
OH
COO-
-OOC
+
NH3
9.54
pseudosubstrate
OH
=O PO
3
N
H
9.55
quinonoid form observed
at 490 nm
Stereochemistry of Proton Transfer in the First Step
Catalyzed by Many PLP-dependent Aminotransferases
-H is transferred to the CH2 of PMP suprafacially;
therefore one-base mechanism
-2H removed from si-face and delivered to pro-S CH2 of PMP
B
B:
H N
-O
H3 C
2H
R
H
OPO3
N
H
-OOC 2H
COO-
9.56
-O
=
H3 C
H N
N
B:
-OOC
R
H
H N
-O
OPO3
=
R
2H
pro-S
H
H3C
OPO3=
N
H
H
H2O
Scheme 9.40
H2N
R
-OOC
O
-O
H3 C
H
OPO3=
N
H
2H
9.57
Cis-Trans Isomerization
Reaction catalyzed by maleylacetoacetate isomerase
COO-
GSH
-OOC
COOO
O
9.58
COOO
O
9.59
Scheme 9.41
GSH acts as a coenzyme, not as a reducing agent
No 2H incorporated into substrate or product from 2H2O
Proposed Mechanism for the Reaction Catalyzed
by Maleylacetoacetate Isomerase
GS
COO-
GS
COO-
-OOC
COOO
O
Scheme 9.42
-OOC
SG
COOO
O
COO-
COOO
O
O
O
Phosphate Isomerization
Reaction catalyzed by phosphoglucomutases
OPO3=
O
HO
HO
OH
OH
9.66
only -anomer binds
Scheme 9.45
HO
HO
OH
O
OH
9.67
OPO3=
Native State of Enzyme is Phosphorylated
Proposed mechanism for the reaction catalyzed
by phosphoglucomutases
B H
Ser O
O
B
32
P
OO
O-
O
HO
HO
OH
9.67
H
Ser O
:B
tightly
bound
OPO3=
H
Ser O PO3=
O32PO3=
O32PO3=
O
HO
HO
9.68
O
OOH
O P O-
B H
HO
HO
O
Scheme 9.46
Overall retention of configuration at phosphate
Double inversion
Shown as associative, but could be dissociative
OH
9.66
OH
Model Reaction for a Dissociative
Mechanism of Phosphomutases
S
SO
18O-
P
O
18O-
P
H
O
O
O
OH
OH
NO2
NO2
solvent
cage
Scheme 9.47
S 18OP
O
O
NO2
~ 40% retention