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The Organic Chemistry of Enzyme-Catalyzed Reactions
Chapter 9
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
Isomerizations
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 2H2O
Therefore deprotonation/reprotonation mechanism
[1,1]-Hydrogen Shift
Racemase with no cofactors
Glutamate racemase
Scheme 9.1
[1,1]-Hydrogen ShiftAmino acid racemases
One base: substrate proton transferred to product
(A) One-base mechanism for racemization (epimerization), (B) Two-base mechanism for racemization (epimerization)
R
H
NH3+
COO-
B
R
NH3+
-OOC
B
R+H3N
-OOC
B
H
R
H
NH3+
COO-
B BH B B
R
NH3+
-OOC
H
R+H3N
-OOCH
BH B
Ha
ab
H
b
A
B
also, primary kinetic isotope effect with [2-2H]GluWith Glu racemase: solvent deuterium in product, not substrate
Two base: incorporated proton from solvent(B)
Figure 9.1
in D2O
An “Overshoot” Experiment with (R)-(-)-glutamate to Test for a Two-base Mechanism for Glutamate Racemase
0
20
60
100
-20
-60
-100
Time (sec)1000 2000 3000
Scheme 9.2
Another Test for a Two-Base MechanismElimination of HCl from threo-3-chloroglutamic acid by the C73A and C184A mutants for glutamate racemase
COO-
COO-
Cl
NH3+
H
H
COO-
-OOC
Cl
NH3+
H
H
SS
COO-
COO-
Cl
NH3+
H
H
COO-
-OOC NH3+
HCOO-
COO-+H3N
H
COO-
-OOC
Cl
NH3+
H
H
C184A
COO-
-OOC O
HD
COO-
COO-O
H
C73A
9.1
D
9.2 9.2
S RS R
SS
D2O
RR
D2O
C73C184
Scheme 9.3
Inactivation by ICH2COO- only after a reducing agent is added (RSH or NaBH4)
Proposed Mechanism for Proline Racemase
HN
H HN
S
HOOC
S
H S
S
S
SH
HN
H
S
S
H
9.3
HO
HOO
HO
‡δ
HN
O
HOδ
H
H
Reduces active site disulfide to dithiol
Transition State Analogue Inhibitor
HN-OOC
9.4
Because substrates bind tightest at the transition state of the reaction, a compound resembling the TS‡ structure would be more tightly bound
TS‡ analogue inhibitor for Pro racemase
resembles 9.3
Scheme 9.4
Pyridoxal 5-Phosphate (PLP) Dependent Racemases
NH
NH
CCH3
H
COO-
NH
NH
CCOO-
CH3
NH
NH
CCH3
COO-
H
CH3
OH OH
CH3
OH
CH3
:B
=O3PO
+Keq ~ 1
L-Ala D-Ala
=O3PO =O3PO
+
+
NH3
CCH3
H
COO-
+ +
+
PLP
+NH3
CCH3
COO-
H
BH
quinonoid intermediate
PLP
PLP
Proposed mechanism for PLP-dependent alanine racemase
Usually, a one-base mechanism
How can PLP enzymes catalyze selective bond cleavage?
PLP was a coenzyme for decarboxylases (break C-COOH bond) and now for racemases (break C-H bond)
Stereochemical Relationship Between the -Bonds Attached to C and the p-Orbitals of the -System
in a PLP-Amino Acid Schiff Base
Figure 9.2
NH
NH
H
R
-OOC
The -bond that is parallel to (overlapping with) the p-orbitals will break (C-H in this case)
PLPall sp2 + p atoms
Figure 9.3
Dunathan Hypothesis for PLP Activation of the Bonds Attached to C in a PLP-Amino Acid Schiff Base
C
RH
COO-
C
H-OOC
R
C N CH
H
N CHN CH
R COO-
+
+
BA C
pyridine ring of PLP
The -charge stops free rotation, which results in selective bond cleavage
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.
Scheme 9.5
No internal return in either direction
Other Racemases
Ph CO2-
HO H
Ph CO2-
H OH
S-mandelateR-mandelate
Reaction catalyzed by mandelate racemase
With (R)-mandelate no -H exchange with solventWith (S)-mandelate there is exchange with solvent
Scheme 9.6
solvent exchange
no solvent exchange
H297N mutant is capable of exchanging the -H of the S-isomer, but not the R-isomer
A Two-base Mechanism for Mandelate Racemase that Accounts for the Deuterium Solvent Exchange Results.
166Lys ND2
Ph
HOH
166Lys N
H
D
D
PhO-
OH
OO-
O
Ph
HO H
ND
N
297HisND
N
297His
H
PhO-
OH
O
O-
OPh
HO H
O-
O
Ph
DOH
O-
O
+
Mg2+
Mg2=
Mg2+ Mg2+
(S)
(R)
A
B
Lys-166 acts on the (S)-isomer, and His-297 acts on the (R)-isomer
Scheme 9.7
H297N Mutant Capable of Elimination of HBr from (S)-9.5, but not from the (R)-isomer
K166R mutant catalyzes elimination of HBr from the (R)-isomer, but not from the (S)-isomer
Elimination of HBr from (S)-p-(bromomethyl)mandelate, catalyzed by the H297N mutant of mandelate racemase
Br
NH2Lys-166
COO-
OH
COO-
O
9.5 9.6
COO-
OHH
-HBr
EpimerasesPeptide epimerases
Scheme 9.8
Mechanism 1
With 18O in the Ser OH group, no loss of 18O as H218O
Elimination/addition (dehydration-hydration) mechanism for peptide epimerization
NH
OH
O
Phe-Ala-OH
O
AcNH-Gly-Leu
H
:B
NH
O
Phe-Ala-OH
O
AcNH-Gly-Leu
NH
OH
O
Phe-Ala-OH
O
AcNH-Gly-Leu
H
BH
BH
NH
OH
O
Phe-Ala-OH
O
AcNH-Gly-Leu
H:B
BH
NH
OH
O
Phe-Ala-OH
O
AcNH-Gly-Leu
H:B
BH
9.7
BH
-OH
Therefore, mechanism 1 is unlikely.
Scheme 9.9
Mechanism 2
10 mM NH2OH has no effect on product formation
NH
O
O
Phe-Ala-OH
O
AcNH-Gly-Leu
H
:B
NH
OH
O
Phe-Ala-OH
O
AcNH-Gly-Leu
H
BH
NH
O
Phe-Ala-OH
O
AcNH-Gly-Leu
H:B
B
H
HH
H
O
9.8
-Cleavage Mechanism for Peptide Epimerization
Therefore, mechanism 2 is unlikely.
Scheme 9.10
Mechanism 3
In D2O D is incorporated into product, not substrate (short incubation; monitored by electrospray ionization mass spectrometry)
NH
OH
O
Phe-Ala-OH
O
AcNH-Gly-Leu
H
B
NH
HO
O
Phe-Ala-OH
O
AcNH-Gly-Leu
H
BH
NH
OH
O
Phe-Ala-OH
O
AcNH-Gly-Leu
H:B
BH
BH
BH
BH
B:
Deprotonation/Reprotonation Mechanism
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.
OHO
H
CH3
H
OdTDPOH
HH
OHH
B+ H O
O
H
CH3
H
OdTDPOH
HH
OH
N
H H
NH2
O
R
9.9 9.10
NADPH
epimerase reductase
O
O
CH3
HH
OdTDPOH
HH
OHB+ H
:B
B+ H
B:
OCH3
H
H
OdTDPOH
HOH
H
O
OO
CH3
HH
OdTDPOH
HH
OH
B:
B+ HO
O
H3C
H
H
OdTDPOH
HH
OH
B+ H
B:
Scheme 9.11
Epimerization with Redox Catalysis
two different enzymes
C-H cleavage at C-3 and C-5 show kinetic isotope effects (3.4 and 2.0, respectively)
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)
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
UDP-glucose UDP-galactose
No change in oxidation state, but is deprotonation/reprotonation reasonable?
OOH
OH
O UDPOH
OH
OOH
OH
O UDPOH
HO
9.11 9.12
In H218O, no incorporation of 18O into product
Scheme 9.12
The enzyme requires NAD+; no exchange with solvent
reverse reaction
without OH
proposed intermediate
Tritium is incorporated from NAD3H into a derivative of the suspected intermediate of the UDP-glucose 4-epimerase-catalyzed reaction
OOH
CH3
O dTDPOH
OO
OH
CH3
O dTDPOH
HO
3H
9.13
E•NAD3H +
Scheme 9.13
Evidence for 9.14: incubate enzyme with UDP-galactose,quench with NaB3H4. 3H at C-4 of both UDP-glucose and UDP-galactose
Proposed Mechanism for Reaction Catalyzed by UDP-Glucose 4-Epimerase
OOH
OH
O UDPOHO
H OOH
OH
O UDPOH
O
B HO
OH
OH
O UDPOH
H
HO
NAD H
+
9.14+
H:B
NAD
NAD
Scheme 9.14
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
OOH
OH
H
O OH
OBH
O
OHOHO
O OH
O
OHO
HO
OH OH
HUDP
UDP UDP
N
H2N O
R
H
:B
N
H2N O
RH
H
-NAD+
Scheme 9.15
No change in oxidation state, but NAD+ required
Mechanistic Pathway for the GDP-D-mannose-3,5-epimerase-catalyzed Conversion of GDP-D-mannose (9.15) to GDP-L-galactose (9.18)
OOH
OH
O GDP
OH
OH
H OOH
OH
O GDP
OHO
O
OH
OH
O GDP
OH
O
OH
O GDP
OHO
9.15OH9.16
O
OH
OH
O GDP
OH
OH
9.179.18
O
H NAD+
NAD+ NADH
NADH
[1,2]-H Shift
Scheme 9.16
Lobry de Brun-Alberda von Ekenstein Reaction
Reaction catalyzed by aldose-ketose isomerases
CHO
C OHH
R
CH2OH
C O
R
9.209.19
Scheme 9.17
Two
Mechanisms
suprafacial transfer of H
cis-Enediol mechanism for aldose-ketose isomerases
C
C O
R
H
OHHBC
C
OH
OR
H
BB:
H
C O
C
R
OHH
B HH
H
:B BH
R
OHHOH
B:
BHOR
OHHB H
H
:B
9.22
OR
*
H
HR OH
*
*
9.21
B
**
B
*
H
(2R)
(2R)
re
re
pro-Rcis-enediol
Mechanism 1
Scheme 9.18
Partial incorporation of solvent observed - inconsistent with hydride mechanism
Hydride transfer mechanism for aldose-ketose isomerases
BH
:BC
C O
R
H
O
H
:B
BH
*
C
C O
R
H
OH
H*
H
Mechanism 2
Scheme 9.19
[1,3]-H Shift
Enolization
removes pro-R hydrogen
Reaction catalyzed by phenylpyruvate tautomerase
CO2-
O
HSHR
R R
HS
OH
CO2-
R = H or OH
Scheme 9.20
Two Conformers PossibleConformations of phenylpyruvate that would form Z- and E-enols by phenylpyruvate tautomerase
CO2-
O
HS HR
H B
B:
CO2-
OH
O
CO2-
HS HR
H B
B:
CO2-
OH
anti
Z
E
syn
favored inhibitors
Therefore syn geometry to E enol most likely
To Test for Favored Conformation
F
CO2-
R CO2-
F
R
CO2-
RCO2
-R
9.23 9.259.24 9.26
Scheme 9.21
Allylic Isomerizations
Carbanion mechanism for allylic isomerases
B:H
B HH
This H could come from the substrate (if no solvent exchange)
Mechanism 1
Scheme 9.22 This H comes from solvent, not from the substrate
Carbocation mechanism for allylic isomerases
B+H
H HH H
B:
Mechanism 2
Scheme 9.23
Unlikely -- [1,3]-hydride shift is allowed antarafacial,which is geometrically impossible
[1,3]-Sigmatropic hydride shift mechanism for allylic isomerases
H H
Mechanism 3
Scheme 9.24
Carbanion Mechanism
Principal reaction transfers 4-H to 6-position; therefore suprafacial
Reaction catalyzed by 3-oxo-5-steroid isomerase
O
H
O
H H
O
H
OHH
12
34
5 6
9.27 9.28(D)
(D)
Eliminates carbocation mechanism and [1,3] hydride shift
Scheme 9.26
Evidence for an Enol Intermediate in the Reaction Catalyzed by 3-Oxo-5-steroid Isomerase
O
O
O
O
O
O
O
HO
9.329.31
9.31
10%
nonenzymaticpH 4.5
9.32
9.349.33(90%)
+
enzymatic
enzymatic
Scheme 9.27
Kinetic Competence of Enol
same rates
Further evidence for an enol intermediate in the reaction catalyzed by 3-oxo-5-steroid isomerase
O
HO
O
O
O
O9.35 9.36 9.37
from NOE studies
From Site-directed Mutagenesis, Tyr-14 is the Acid and Asp-38 the Base
O
H
H
Asp-38
O O
OH Tyr-14
OH Tyr-14
OH Tyr-14
9.38
suprafacial
orthogonal (favored)
antarafacial
To probe the function of Tyr-14
Scheme 9.28
Uv spectrum bound to enzyme is same as neutral amine.
Structure bound to enzyme even at low pH (pKa of the phenol must be very low).
Reactions Designed to Investigate the Function of Tyr-14 at the Active Site of 3-oxo-5-steroid Isomerase
H2N
OH
H3N
OH
HO
O
-O
O
9.39
9.40
- H+
+ H+
+
Therefore Tyr-14 does not protonate C-3 carbonyl
Therefore Tyr-14 H bonds to dienolate
Scheme 9.29
Carbanion Mechanism
Mechanism for suprafacial transfer of the 4-proton to the 6-proton of steroids catalyzed by 3-oxo-5-steroid isomerase
O
O
O
O
O
O
H2H HOH
Tyr-14
COO-
Asp-38
O
Tyr-14
HCOO
Asp-38
2HO
H
Tyr-14
2H H
COO-
Asp-38
_
Asp-99 Located Adjacent to Tyr-14
Scheme 9.30
One mechanism for the function of Asp-99 in the active site of 3-oxo-5-steroid isomerase
O
H H
O
HOTyr14
HCOOAsp99
O
OH
O
HOTyr14
HCOOAsp99
OH
O
HOTyr14
HCOOAsp99
OO
38Asp
H H
38Asp
O OO
38Asp
H
equilenin
Crystal structure with equilenin bound is consistent with Asp-99 and Tyr-14 both coordinated to oxyanion
HO
O
9.41
4-Oxalocrotonate Tautomerase
O
CO2-
CO2-
OH
CO2-
CO2-
O
CO2-
CO2-
9.42 9.43 9.44
Scheme 9.32
From deuterated substrates, substrate analogues, and reactions run in D2O, 9.42 to 9.44 is suprafacial(one-base mechanism)
Scheme 9.33
Carbocation Mechanism
No exchange of solvent into substrate, only into product
Reaction catalyzed by isopentenyl diphosphate isomerase
P
O
O-
P
O
O-
O-
9.469.45
OO P
O
O-
P
O
O-
O-OOMg++
isopentenyl diphosphate dimethylallyl diphosphate
One base mechanism
rate is 1.8 10-6 times Ki = 14 pMOP2O6
3-
CF3 OP2O63-
HN+
OP2O63-
9.48 9.49
Evidence for a Carbocation Mechanism
transition state analogue inhibitor
Scheme 9.35
Proposed Mechanism for Isopentenyl Diphosphate Isomerase
OPP
B 2H
OPP
H
B:
OPP2H
2H
Scheme 9.36
Aza-allylic Isomerization
+NH
H
+NH
H
Scheme 9.37
PLP-dependent
AminotransferaseReaction catalyzed by L-aspartate aminotransferase
-OOC CH214C
H
COO-
NH3+
13C COO-
O
-OOCCH214C
18O
COO- CH313C
H
COO-
15NH3+
15
+CH3+H2
18O
Scheme 9.38PMP
First Half Reaction Catalyzed by Aspartate Aminotransferase
NH
NH
OH-OOC 14C COO-
H
15NH2
-OOC 14C COO-
H
15NH
NH
OH
B:
-OOC 14C COO-
15NH
NH
OH
B H
-OOC 14C COO-
15NH
NH
OH
-OOCCH214C
18O
COO-
15NH3
NH
OH
9.50
aldimine
=O3PO=O3PO
=O3PO
=O3PO
=O3POslow step
quinonoid
9.51
9.52
..
H218O
see Scheme 8.39
Scheme 9.39
Second Half Reaction Catalyzed by Aspartate Aminotransferase
CH3 13CCOO-
15NH
NH
OH
B15NH3
NH
OH13C COO-
O
CH3 HB:
CH3 13CCOO-
15NH
NH
OH
HCH3 13C
COO-
15NH
NH
OH
H
NH2
NH
NH
OH
CH3 13CCOO-
15NH3
H
=O3PO
=O3PO=O3PO
=O3PO
=O3PO
9.53
9.52
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
pseudosubstrate quinonoid form observed at 490 nm
NH
OH
NH
COO--OOC
OH
NH3
COO--OOC
OH
9.54
+
+
=O3PO
9.55
Evidence for Quinonoid Intermediate
Scheme 9.40
-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
Stereochemistry of Proton Transfer in the First Step Catalyzed by Many PLP-dependent Aminotransferases
N
-O
H
B:
N
2H
H
H3C OPO3=
H
COO-
R
N
-O
H
B
N
2H
H
H3C OPO3=
H
-OOC R
N
-O
H
B:
N2H
HH3C OPO3
=
H
-OOC R
N
-O
H
H2N2H
H
H3C OPO3=
-OOC R
O
9.56
9.57
H2O
pro-S
Scheme 9.41
Cis-Trans Isomerization
GSH acts as a coenzyme, not as a reducing agent
No 2H incorporated into substrate or product from 2H2O
Reaction catalyzed by maleylacetoacetate isomerase
COO-
COO-
O O
COO-
O O
-OOCGSH
9.58 9.59
Scheme 9.42
Proposed Mechanism for the Reaction Catalyzed by Maleylacetoacetate Isomerase
COO-
COO-
O O
COO-
O O
-OOCCOO-
COO-
O O
GS SG
COO-
O O
-OOCGS
Scheme 9.45
Phosphate Isomerization
only -anomer binds
Reaction catalyzed by phosphoglucomutases
O
OHOH
HO
O
OPO3=
HO
OPO3=OH
HO
OH
HO
9.679.66
Scheme 9.46
Native State of Enzyme is Phosphorylated
tightly bound
Shown as associative, but could be dissociative
Proposed mechanism for the reaction catalyzed by phosphoglucomutases
O
OHOHHO
HOO
OPO3=OHHO
O
HO
P
O
O-O O-Ser
B H
Ser O PO3=Ser O
PO-
O
O-
O
OOHHO
O32PO3=
HO
B H
O32PO3=
B32 H
H:B
9.689.67 9.66
Overall retention of configuration at phosphateDouble inversion
Scheme 9.47
Model Reaction for a Dissociative Mechanism of Phosphomutases
NO2
OP
O
18O-S-
OH
NO2
OO
NO2
OHO
PO
18O-S
solventcage
~ 40% retention
PO
18O-S
H