Transcript The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 10 Eliminations and Additions Anti Eliminations and Additions Reactions catalyzed by dehydratases and hydratases H R C R' H C R'' OH Scheme 10.1 -H2O R +H2O R' C CHR''

The Organic Chemistry of
Enzyme-Catalyzed Reactions
Chapter 10
Eliminations and Additions
Anti Eliminations and Additions
Reactions catalyzed by dehydratases and
hydratases
H
R
C
R'
H
C
R''
OH
Scheme 10.1
-H2O
R
+H2O
R'
C
CHR''
Three General Mechanisms for Dehydration
(nonenzymatic)
B
C
C
H
E1cB
C
fast
OH
C
C
slow
OH
+
carbanion
mechanism
OH
C
stabilized carbanion
B
C
HO
H
C
E2
C
+
C
concerted
mechanism
H2O
B
C
H
E1
C
H
C
OH
fast
C
OH2
H
B
slow
C
C
H
C
C
carbocation
mechanism
Scheme 10.2
Enzymatic Dehydrations
When H is next to COOH, anti-dehydration
When H is next to aldehyde, ketone, or thioester,
syn-dehydration
M2+-dependent Dehydration
Reaction catalyzed by enolase
Scheme 10.3
COOOPO3=
H
HA
HB
OH
10.1
2-phospho-Dglyceric acid
(2-PGA)
Kd ~ 1
-OOC
OPO3=
-H2O
+H2O
HA
5.33 ppm
in NMR
HB
10.2
phosphoenolpyruvate
(PEP)
2 Mg2+ required
5.15 ppm
in NMR
Anti- versus Syn-Elimination of Water
from 2-Phosphoglycerate (PGA)
H
2H
H
anti
-OOC
OPO3=
-H2O
+H2O
-OOC
C
2H
OPO3=
C
H
OH
(3R)-[3-2H]-2-PGA
H OH
-OOC
syn
C
H
-OOC
Scheme 10.4
OPO3=
2H
OPO3=
C
H
2H
NMR 5.14 ppm;
therefore antielimination
Stereochemistry of Water Addition to
Phosphoenolpyruvate Catalyzed by Enolase
Back reaction
H
si
COO-
+H2O
H
re
OPO3=
-H2O
H
H
H
HO
COO2R
PGA
Scheme 10.5
OPO3=
proton adds to
si-face of PEP;
therefore OH must
add to re-face
Relative Rates of Exchange in the
Enolase-catalyzed Reaction
krel
14PGA
14PEP
[3-18O]PGA
[2-2H]PGA
H218O
2
H2O
1.0
1.3
1.9
slow step release of PEP
fast step deprotonation
therefore E1cB
Evidence for E1cB Mechanism for Enolase
3H
exchanges into 2-PGA
B 3H
3H
2O
fast
M2+
B H
O
C
B:
M2+
O-
O
O
H
C
OPO3=
H
C
H
C
H
C
B+
H
H
10.3
aci-carboxylate
Scheme 10.6
-OOC
slow
OH
OH
2-PGA
OPO3=
C
H2O
+
OPO3=
C
CH2
Evidence for Aci-carboxylate
Intermediate (10.3)
O
O
N
PO3=
O
HO
O
PO3=
O
O
N
PO3=
O
HO
HO
10.4
10.5
10.6
All are potent inhibitors
M2+
O
OPO3=
C
O
C
H
C
OH
10.3
H
PO3=
NH
10.7
Crystal Structure at 1.8 Å
Resolution of Yeast Enolase
Schematic of the yeast enolase
active site showing the
coordination of the residues and
the substrate to the two Mg2+ ions.
The dashed lines from the 2-PGA
to amino acids represent possible
hydrogen bonds. The dashed lines
from the Mg2+ ions indicate their
coordination. Interatomic distances
in angstroms are given on the
dashed lines.
Mg2+
Mg2+
Mg2+
Mg2+
O
C
O
Figure 10.1
HO
O
H
P
O
C
H
O
211Glu
O
O O
CH2
NH2
Mg2+ coordination lowers
pKa of the C-2 H+
Lys-345 is in a hydrophobic
region - lowers pKa,
increases free base form
Lys345
NAD+-Dependent Elimination
Reactions catalyzed by nucleoside diphosphohexose-4,6dehydratases (oxido-reductases). NDP stands for
nucleoside diphosphate. The sugar positions are numbered.
oxidized reduced
4
HO
HO
6
OH
O
5
3 HO
10.11
NAD+
2
O
OH
NADH
1
ONDP
O
O
HO
O
NADH
O
NAD+
HO
HO
OH
ONDP
10.12
HO
ONDP
10.13
CH3
O
HO
10.14
ONDP
Scheme 10.8
• dTDP-[4-3H]10.11
dTDP-[6-3H]10.14 (intramolecular)
• In 2H2O product incorporates 2H at C-5
• All 3H released from dTDP-[5-3H]glucose
• With [4-3H]NAD+ no 3H in product (suggests intramolecular)
Test for Intramolecular or Intermolecular H Transfer
4
HO
HO
6
OH
O
5
3 HO
10.11
NAD+
2
1
ONDP
O
OH
NADH
O
O
HO
O
NADH
HO
HO
ONDP
10.12
OH
NAD+
O
HO
ONDP
10.13
CH3
O
HO
10.14
ONDP
Crossover experiment: labeled and unlabeled substrates added together
and look for transfer of atom or group to other substrate
If this occurs, then intermolecular transfer
Mixture of dTDP-10.11 + dTDP-10.11-d7 gives only dTDP-10.14
and dTDP-10.14-d6; therefore no crossover
C-4 transferred to C-6 intramolecularly
Proposed Mechanism for the Reactions Catalyzed by
Nucleoside Diphosphohexose-4,6-dehydratases.
The C-4 hydrogen is labeled and the solvent is D2O so the results of the
experiments described above are apparent
R
D2 N
H
H
D2N
H
O
3H
OD
O
DO
D2O
ONDP
10.11
Scheme 10.9
B
DO
DO
B:
H
B
D2 N
H
O
D2 N
H
DO
D
O
O
3H
H
O
of H- and H+
D2N
3H
O
O
D
B:
OD
D
ONDP
B: 10.14anti-addition
H
H
H
H
suprafacial 3H
transfer
N
O
3H
OD
ONDP
washed out
R
N
B
O
D
OD
H
ONDP
10.12
D
OD
R
N
B
B
O
B:
R
O
D
3H
O
O
O
D
OD
B:
D2N
3H
O
OD
O
D
N
N
N
antielimination
of H2O
R
R
DO
OD
ONDP
D
B
O
D
B
DO
B
OD
D 10.13 ONDP
Determination of the Stereochemistry of Me Groups
Transfer of the C-4 hydrogen of (6S)-10.15 and (6R)-CDP[4-2H, 6-3H]D-glucose (10.17) to the C-6 methyl group in the
CDP-4-keto-6-deoxyglucose product
chirally tritiated
2H
HO
3H
H
O
CDP-D-glucose 4,6-dehydratase
O
HO
HO
chiral Me group
HO
OH
OCDP
10.15
HO
H
3H
O
HO
HO
O
CDP-D-glucose 4,6-dehydratase
HO
OH
10.17
OH
OCDP
10.16
epimeric
Me group
chirally tritiated
2H
CH2H3H
O
OCDP
Scheme 10.10
CH3H2H
O
OH
OCDP
10.18
Kuhn-Roth Oxidation of
CDP-4-keto-6-deoxyglucose
O
HO
CH2H3H
O
30% aq. CrO3
OH
OCDP
10.16
Scheme 10.11
H2SO4
155 oC
T
D
D
COOH
H
or
T
COOH
H
S
R
Polarimetry will not work;
3H only in trace amount
Enzymatic Conversion of Chiral Methyl-containing Acetate into
Fumarate for Determination of the Chirality of the Methyl Group
get both products
(only detecting 3H products)
2S-malate
2S
T
T
1. acetate kinase/ATP
D
D
COOH
2. phosphotransacetylase/CoASH
S
Scheme 10.12
H
O
HOOC
H
1. malate synthase/glyoxylate
SCoA 2. hydrolysis
H
OH
COOH
OH
COOH
COOH
T
antielim.
H
HOOC
COOH
T
10.20
-3H2O fumarase
D
H
10.19
free rotation
OH
OH
+
T
D
H
HOOC
-H2O
fumarase
H
HOOC
+
HOOC
H
T
10.19
HOOC
H
D
H
10.20
(KIE = 3.8)
COOH
10.21
80%
With 10.16: 71% T in fumarate; therefore (R)-acetate
With 10.18: 30% T in fumarate; therefore (S)-acetate
supports inversion of stereochemistry
T
COOH
10.22
20%
Outcomes of the Malate Synthase/Glyoxylate
Reaction Followed by Hydrolysis
H
HOOC
T
O
O
O
D
T
SCoA
H
HOOC
HOOC
OH
COSCoA
D
OH
T
D
B:
COOH
10.19
HOOC
H
T
O
H
SCoA
D
H
O
O
B
H
HOOC
H
OH
HOOC
H
T
SCoA
T
H
OH
hydrolysis
H
COSCoA
T
B:
COOH
10.20
HOOC
D
H
O
D
SCoA
T
B:
H
hydrolysis
T
SCoA
D
H
B
H
H
No 3H; not detected
H
O
O
B
H
HOOC
H
OH
D
SCoA
H
HOOC
H
OH
hydrolysis
D
COSCoA
H
COOH
Slide not in text--after Scheme 10.12
Iron-sulfur Clusters in a Nonredox Role
Aconitase-catalyzed interconversion of citrate
(10.23) and isocitrate (10.25) via cis-aconitate
COOH
H
-OOC
COO-
dehydration
OH
H
H
-OOC
H
-H2O
+H2O
+H2O
-OOC
CH2
COO-
COO10.23
citrate
Scheme 10.13
hydration
10.24
cis-aconitate
-H2O
H
OH
-OOC
H
H
H
COO10.25
isocitrate
(2R, 3S)
Citrate is Prochiral
removed in going to cis-aconitate
COOpro-R arm
HS
HR
-OOC
OH
HR
HS
COO10.26
pro-S arm
Stereochemistry of Elimination of Water
from Citrate Catalyzed by Aconitase
Scheme 10.14
H
OH
-OOC
B
HS
-OOC
H
2
3
-OOC
B:
HR
10.23
COO-
-OOC
COO10.24
must be anti-elimination
to give cis-aconitate
Stereochemistry of Addition of Water to Cisaconitate to Give Citrate (back reaction)
(re-si)
HO
-OOC
COOsi
re
2
H
-OOC
OH
H
H2O
-OOC
anti-addition
3
COO10.24
H
(si-re)
Scheme 10.15
H
10.23
COO-
Stereochemistry of Addition of Water to
Cis-aconitate to Isocitrate
(re-si)
H+
-OOC
re
COO-
H2O
si
2
3
H
COOH
anti-addition
-OOC
-OOC
HO
-OH
(si-re)
cis-aconitate
H
10.24
COO-
10.25
isocitrate (2R, 3S)
Scheme 10.16
Therefore C-2 is always attacked on the
face opposite attack at C-3
Labeling studies show that the pro-R proton removed
from C-2 of citrate ends up at C-3 of isocitrate!
Overall Stereochemistry of the Aconitasecatalyzed Reaction
HO-
citrate
-H2O OOC
COO-
H
*H+
(si-re)
Scheme 10.17
HO-
COOflip
COOH2O
-OOC
H
*H+
COO-
(re-si)
isocitrate
A Crossover Experiment with Aconitase in Which
[(2R)-3H]citrate and 2-Methyl-cis-aconitate (10.27)
Produce Unlabeled Cis-aconitate and 2-Methyl-[33H]isocitrate (10.28)
COO
COOH
-OOC
C
3H
C
OH
CH2
COO-
-OOC
CH3
aconitase
+
-OOC
10.27
-OOC
H
+
C
-OOC
C
OH
3H
CH2
-OOC
COO-
H3C
COO-
COO10.28
crossover product
Scheme 10.18
observed
Therefore the proton removed from one substrate molecule
can be transferred to a different substrate molecule
(intermolecular)
The OH exchanges with solvent, but the proton removed does not!
A Proposed Mechanism for Aconitase
B:
+ 3
B H
3H
OH
H
H
-OOC
X
COOCOO-
18OH
18
OH
+
B
-OOC
3H
COO-OOC
X
18
OH
COO-
COOH
flip
(after release
from active site)
X
OH
COO-
Scheme 10.19
3H
B:
-OOC
H
X
COOCOOOH
Where does the iron-sulfur cluster come in?
protein
S
Fe
Fe
protein
protein
Fe
S
S
S
H
-O
O
Fe
H
H
O
H
O
O
O
COO-
from crystal
structure
H
COO-
C
165Asp
10.29
Fe acts as a Lewis acid - nonredox role
Support for Aci-carboxylate Bound
to Fe-S Cluster
very potent
inhibitor
COOO
O
H
OH
N
H
H
H
COO-
COOO
very
acidic
10.30
product mimic
O
H
OH
C
H
H
H
COOisocitrate
COOO
H
OH
COOO
N
O
H
OH
C
H
H
COO10.31
O
H
H
COO10.32
Crystal structure with 10.31 bound is same
as with isocitrate bound (to Fe-S cluster)
Therefore, ElcB (carbanion) mechanism
Elimination of Phosphate
Reaction catalyzed by chorismate synthase
COO-
COOHR
2
3
=O PO
3
1
4
6
HS
antielimination
HS
+
5
O
O
COO-
OH
OH
10.39
10.40
EPSP
PO43-
+
HR+
COO-
chorismate
Scheme 10.22
Orbital symmetry rules: concerted 1,4-elimination is
syn - suggests stepwise elimination
=O PO
3
COO-
O
COO-
OH
10.41
not a substrate
Therefore not [1,3]-rearrangement of phosphate
COO-
=O PO
3
COO-
COO-
:B
H
=O PO
3
O
OH
O
COOOH
10.41
O
COO-
OH
COO-
Other Possibilities
E1 (pathway a) and addition/elimination (pathway
b) mechanisms for chorismate synthase
COO-
:B
H
H
+
O
E1
-
b
OH
a
X
COO-
- PO43-
COO-
COO-
H
H
=O
3PO
O
a
O
COO-
OH
OH
E2
b
c
- PO43-
covalent
X
COO-
:B
-X
H
O
OH
:B
COOH
E1
d
H
Scheme 10.23
COO-
H
O
COOOH
COO-
To Test These Mechanisms
COORR
F or H
RS
=O PO
3
O
COO-
OH
10.42
Neither was a substrate nor an inactivator
Covalent mechanism would give inactivation when RR = F
(by either b-d or b-c)
Consistent with E1
However, a flavin is required; Fl-. observed in EPR
Radical Mechanism Proposed for
Chorismate Synthase
CO2-
CO2-
CO2-
O
H
CO2-
OH
+ e-
-H
=O
3PO
O
CO2-
CO2-
=O
OH
3PO
O
OH
CO2-
CO2-
O
OH
O
OH
Scheme 10.24
CO2-
CO2-
Chemical Model in Support of the Radical
Mechanism for Chorismate Synthase
CO2Me
CO2Me
Br
SnBu3
O
(PhO)2 P O
O
CO2Me
-(PhO)2PO2
(PhO)2 P O
OTBDMS
Scheme 10.25
OTBDMS
OTBDMS
Elimination of Ammonia: Ammonia Lyases
Reaction catalyzed by histidine
ammonia-lyase (HAL)
D
D
D
COO-
N
NH3+
D
N 5'
H
[D3]His
HAL
H2O
COOH
N
3
N
H
2
1
H
10.43
urocanic acid
dehydroalanyl-dependent
Scheme 10.26
Evidence for Dehydroalanyl Enzyme
Reactions to identify the active-site prosthetic group
as a dehydroalanyl moiety
O
NaB3H
4
3HH C
2
H3O+
NH
3HH
COOH
2C

NH3+
NH
Ala
O
O
14CN
NH
H3O+
N14C
NH
NH

COOH
HOO14C
NH3+
NH
10.44
Asp
14CH
2NO2
O2N
14CH
2
O
NH
NH
Scheme 10.27
COO-
1. H2/Pt
2. 6 N HCl

14
+NH
NH3+
3
10.45
Dbu
Actual Prosthetic Group is Not
Dehydroalanyl, but Something Related
Posttranslational conversion of the active site
Ala-Ser-Gly at positions 142-144 to give a
dehydroalanyl-like species
N 142
H
H
N 143
O
H
N
H
N
O
..
N 144
H
OH
N
H
H
N
O
Ala-Ser-Gly
B
N
N
H
O
N
O
H
OH
N
N
H
O
O
B
10.45a
crystal structure
Scheme 10.28
Stereochemistry of the Elimination
Catalyzed by Histidine Ammonia-lyase
HS
3H
R
H
COOH
N
H
N
H
NH3+
-3HR+
-NH3
COOH
N
N
H
Scheme 10.29
In 3H2O 3-[pro-R] hydrogen of His is exchanged
(lost in conversion to urocanate)
(anti-elimination)
His + [2-14C]urocanate  [14C]His (reversible)
urocanate + NH3  His (reversible)
Initial Proposed Mechanism for Histidine
Ammonia-lyase
H
COO-
N
NH2
:
N
H
H
O
B
NH
COO-
N
N
H
NH
:B
NH2 O H
NH
NH
H B
:B
H
COO-
N
N
H
COO-
N
NH2 O
COO-
N
NH2 O
N
H
NH
NH
N
H
NH3 O
NH
NH
NH
H
D2O
Scheme 10.30
NH
COO-
N
N
H
D
-NH3
NH2 O
O
NH
NH
What’s wrong with this mechanism?
The pKa of the proton being abstracted is very high.
NH
NH
B
Activation of C-3 Deprotonation by a 2-Nitro
Group in the Histidine Ammonia-lyase Reaction
O
O
N
N
H
O
B
H
COONH NH2
O
N
N
COONH NH2
Scheme 10.31
This is a good substrate even with mutants
that do not contain dehydroalanyl-like group
Alternative Role for the Prosthetic Group
Proposed alternative (electrophilic aromatic substitution)
mechanism for histidine ammonia-lyase
makes the C-3 proton more acidic
N
H
N
H
N
N
H
B
:B
N
H
N
N
O
H
H
O
N
:B
O
H
H
H
COO-
N
: NH NH3
electrophilic
aromatic
substitution
N
H
-NH3
H
COO10.45a
:B
N
COO-
NH NH3
N
10.46
:NH NH3
10.47
N
H
:B
N
N
N
H
O
N
N
H
H
B
O
10.45a
COO-
Scheme 10.32
H
COON
NH
N
10.48
NH
Syn-Eliminations and Additions
Reaction catalyzed by 3-dehydroquinate
dehydratase (3-dehydroquinase)
Scheme 10.33
HO
HR
COO-
COOsyn-elimination
HS
H
+
O
OH
O
HR
OH
OH
OH
10.49
10.50
3-dehydroquinate
3-dehydroshikimate
NaBH4 inactivates the enzyme in the presence of substrate
One 3H incorporated into protein with NaB3H4 + substrate
Schiff Base Mechanism
Proposed mechanism for 3-dehydroquinate
dehydratase (3-hydroquinase)
B
HO
COO-
B:
HO
HR
HR
HS
HS
Lys
H
HO
OH
H2O
HS
+
HN
OH
COO-
COO-
COO-
+
O
NH2
COO-
OH
HN
OH
OH
Lys
detected by
electrospray MS
OH
HN
OH
NH2
Lys
Lys
NaBH4
COO-
Scheme 10.34
HN
OH
OH
Lys
OH
OH
OH
ElcB
O
Lys
PLP-dependent Eliminations
Pyridoxal 5-phosphate-dependent -elimination
(A) and -elimination (B) reactions
X
A
R CH CHCOO-
E•PLP
RCH C COO-
NH3+
B
H2 O
RCH2 C COO-
NH3+
X CH2 CH2 CHCOO-
E•PLP
NH3+
Scheme 10.35
+ NH4+
O
CH3CH C COONH3+
H2O
-elimination
CH3CH2 C COO- + NH4+
O
-elimination
Pyridoxal 5-Phosphate-dependent -Replacement
(A) and -Replacement (B) Reactions
X
A
Y
R CH CHCOO-
E•PLP
Y-
NH3+
B
X
R CH CHCOO-
CH2 CH2 CHCOONH3+
Scheme 10.36
-replacement
NH3+
E•PLP
Y-
Y
CH2 CH2 CHCOONH3+
-replacement
Proposed Mechanism for PLP-dependent
-Elimination Reactions
H
=O
X
D
COO-
3H
+ E•PLP
NH3+
3H
3PO
H N
H3C
H
D
10.52
10.51
..
H N
H3C
COOX HB
N
O
=O
H
=O
3H
-OOC
H
+ E•PLP
D
3PO
3H
: NH
H
N
D
COO-
H
O
10.55
H2O
D
-OOC
H
+
a :NH2
3PO
D
10.56
O
NH3
+
3H
-OOC
H
D
10.56
Scheme 10.37
N
b
O
B
+ E•PLP
3H
H
B
COO-
H
D
10.54
=O
NH3
B
-XH
H N
H3C
3H
H2O
H N
H3C
B
X
b
O
COO- H
10.53
=O
B
H
N
H
O
a
H N
H3C
3H
detected
spectrally
:B
detected spectrally
NH
3PO
detected by
NaBH4 treatment
3H
3PO
N
O
10.57
B
H
H
D
COO-
B
B
Proposed Mechanism for PLP-dependent
-Replacement Reactions
X
H
=O
D
COO-
3H
+ E•PLP
H N
H3C
NH3+
10.51
3H
3PO
N
H
O
D
10.52
=O
H
3PO
H N
H3C
COOX HB
3H
H
N
O
COO-
H
D
10.53
:B
H
B
X
B
-XH
=O
3H
3PO
H N
H3C
H
COO-
N
H
O
D
Y
:B
H
E•PLP
+
3H
Y
D
COONH3+
Scheme 10.38
=O
H N
H3C
3PO
3H
N
O
H
H
COOD Y
B
=O
H N
H3C
3H
3PO
H
N
O
H
10.54
Y
COOD
B
Reaction Catalyzed by Tryptophan
Synthase
3
HOCH2
CHCOO-
+
NH3+
N 2
H
indole
COOPLP
N
H
Trp
NH3+
+ H2O
Scheme 10.39
22 tetramer
 subunits contain PLP - catalyze -elimination part
 subunits needed for  replacement
Proposed Mechanism for Tryptophan Synthase
in the Absence and Presence of  Subunits
B 3H
B:
3H
HO
3H
COO-
B H
HO
COO-
HO
E•PLP
B 3H
COO-
NH+
NH3+
=O
O-
3PO
=O
=O
O-
3PO
NH+
-H2O
NH+
3PO
10.58
=O
O-
3PO
=O
3H
With
2H
COO-
HO
H
NH3+
detected by NMR
O-
3PO
=O
O-
3PO
..
N
H
comes
from -H 3H
2H
COO-
Trp
synthase

H
R
H
NH+
N
H
..
N
H
N
H
10.58
O
pyruvate
COO-
NH+
N
H
COO+ NH4+ + E•PLP
B:
COO-
and indole
or indole
H2 O
H
COOin the presence
of  subunits
in the absence
of a subunits
B+
H
NH+
3H
N
H
B:
..
N
H
O-
..
N
H
N
H+
Scheme 10.40
COO-
N
H
=O
COO-
NH+
E•PLP + Trp
O-
3PO
N
H+
same result in D2O
(still get H incorporated)
O
Suprafacial syn-elimination from si face
Proposed Mechanism for the Reaction Catalyzed by
Tryptophanase
Scheme 10.41
3H
COO-
N
H
E•PLP
+
This is not a
leaving group
COO-
NH+
N
H
COO-
NH+
N
H
O-
=O PO
3
H
COO-
:
NH3
B+3
H
B:
3H
transferred from C-2
This is a
leaving group 3
O-
=O PO
3
NH+
N
H
O-
=O PO
3
..
N
H
10.60
N
H
10.59
N
H+
synelimination
COO-
suprafacial [1,3]H+ transfer
COOB
O
CH3C
COO-
H2O
H3C
COO-
NH2
: NH2
NH2
+ NH4+
E•PLP
H
3H
COOH
NH3+
NH
KIE 3.6
H
NH+
O-
=O PO
3
detected
3H
..
NH2
O-
=O PO
3
N
H
10.61
2H O
2
N
H
H
COO-
3H
+
N
H
N
H
10.58
COO-
2H
O
retention of configuration
Exact reverse of Trp synthase
Stereochemical Differences between Trp
Synthase and Tryptophanase
*
N
H R
S
10.62
COO
*
NH3+
S
S
Have opposite inhibitory potencies with the two
enzymes; therefore opposite stereochemistry
Proposed Difference in the Stereochemistry of the Reactions
Catalyzed by Tryptophanase and Tryptophan Synthase
COOH
NH+
N
H
re,re
=O
O-
3PO
COO-
H
N
H
tryptophanase
N
=O
si,si
O-
3PO
H
COON
H
=O
NH+
O-
3PO
N
H
tryptophan synthase
Scheme 10.42
NH+
:
N
H
N
H
-Elimination and -Replacement
Reactions catalyzed by -cystathionase (A) and
cystathionine -synthase (B)
Scheme 10.43
-cystathionase
NH3+
A
H
S
H
(-elimination)
NH3+
E•PLP
COO-
-OOC
H
NH3+
10.63
SH
COO+
-OOC
-OOC
O
O
NH3
O
cystathionine
B
+
(-replacement)
NH3+
H
COONH3+
10.64
O-succinyl-L-homoserine
NH3+
E•PLP
+
H
-OOC
SH
succinic
acid
H
S
-OOC
H
COONH3+
cystathionine -synthase
Some internal return (not 100%); therefore suprafacial
Proposed Mechanism for the Reaction Catalyzed
by PLP-dependent -Elimination Enzymes
Hc
Hb
=O PO
3
Ha
X
COO- + E•PLP
Hd
H N
H3C
He NH3+
only partial
internal return
He
=O PO
3
H N
H3C
Ha,b,x
N
O
H
X
Hc
N
COO-
H
O
He
Hd
He
Ha
10.65
=O PO
3
H N
H3C
Hb
Hd
Hc
X
N
COO-
H
O
pro-R
Hb
..
HxHaB
10.66
HxB
He
Hd
..
Hc
H N
HxHaHbB
H3C
COO-
=O PO
3
He
Hd
HxHaHbB
Hc
N
COO-
H
O
=O PO
3
H N
H3C
X
..
N
O
H
Hd
HxHaHbB
Hc
COO-
10.67
10.68
10.69
solvent H+
He
=O PO
3
H N
H3C
Hd
N
O
H
10.70
He
Hc
Ha,b,x
Hx
COO-
H2O
Hd
Hc
Ha,b,x
O
+
Hx
NH3
+
E•PLP
Scheme 10.44
COO-
Hx represents solvent protons. HxHbHaB implies that one or more of these
protons is attached to the base B.
Proposed Mechanism for the Reaction Catalyzed by
PLP-dependent -Replacement Enzymes.
Hc
Hb
X
=O
Ha
+ E•PLP
COO-
H N
H3C
He NH3+
Hd
3PO
=O
X
Hc
N
COO-
H
O
He
Hd
He
Ha
10.65
3PO
H N
H3C
Hd
Hc
X
N
COO-
H
O
pro-R
Hb
..
HxHaB
Hb
10.66
HxB
He
=O
..
H N
H3C
3PO
Hd
Y
H
H N
HxHaHbB H3C
COO-
He
..
H N
H3C
3PO
N
O
H
10.72
COO-
Y
Hc
HxHaHbB
3PO
X
..
N
H N
H3C
H
O
10.67
He
Hc
Hx
COOHxHaHbB
=O
H N
H3C
3PO
Hd
COO-
N
H
10.73
Hx
Hx
Hc
HxHaHbB
COO-
Hd
Hc
Y
O
He
Hd
synelimination
solvent H+ (suprafacial)
Hd
Y
N
H
O
=O
10.68
10.71
=O
He
3PO
Hc
..
N
O
=O
He
Hd
Hc
Y
COO-
H3N
+
E•PLP
Hx
Hx
Hx represents solvent protons. HxHbHaB implies that one or more of these
protons is attached to the base B.
Scheme 10.45
Mechanism-based Inactivator of
-Cystathionase
14
COO-
NH3+
10.74
Incorporates 2 mol radioactivity/mol tetrameric enzyme
(half-sites reactivity) with covalent attachment to enzyme
[-2H]10.74 KIE 2.2 on inactivation
Demonstrates removal of C-2 proton for inactivation
Acid hydrolysis of radiolabeled enzyme gives
14
O
COO-
NH3+
10.75
Mechanism-based Inactivation of
-Cystathionase by Propargylglycine
=O PO
3
COONH3+
H N
H3C
+ E•PLP
=O PO
3
H
O
H N
H3C
COO-
N
H
H
H
H
N
COO-
H
O
H
..
HxHB
HxB
H2C
X
=O PO
3
O
H
COO- H HHB
x
H
X
=O PO
3
..
N
H N
H3C
H2C
C
HC
C
=O PO
3
H
H N
N
H3C
O
H N
H3C
HxHHB
COO-
H
..
H N
H3C
X
H2C
C
=O PO
3
=O PO
3
H
..
N
H
O
N
O
H
Hx
COO-
Scheme 10.46
HxHHB
H N
H3C
X
C
H
COOHx
N
O
H
10.77
H3O+
PLP + 10.75
O
Hx
inactivated enzyme
H3N
X
C
10.76
H2C
H
COOH
H
COOHxHHB
Another Mechanism-based Inactivator of
-Cystathionase
F 3C
COONH3+
10.78
2 mol/tetramer
3 F- released/mol inactivator incorporated
max = 519 nm
Denaturation releases all radioactivity as 14CO2
Denaturation in 3H2O incorporates one 3H into
enzyme; hydrolysis gives [3H]Gly
Mechanism-based Inactivation of
-Cystathionase by ,,-Trifluoroalanine
Scheme 10.47
=O PO
3
14COO-
F3C
H N
H3C
+ E•PLP
NH3+
F
F
14COO-
N
H
O
..
H N
H3C
H
F
=O PO
3
F
N
H N
H3C
..
X
F
-F-
HxHB
N
O
H
14COO-
-F-
=O PO
3
..
H N
H3C
F
=O PO
3
F
X
N
O
F
14COO-
H
O
H
HxB
=O PO
3
F
H
14COO-
F
H N
H3C
F
N
X
H
O
14COO-
max 519 nm
F
=O PO
3
..
H N
H3C
X
O
14COO-
H
X
H
X
H N
H3C
N
O
O
=O PO
3
C
H N
H3C
N
H2O
=O PO
3
-F-
14COO-
N
H
O
14COO-
10.79
3H O+
3
O
=O PO
3
COOH
PLP +
H3N
3H
H
[3H]Gly
H3O+
H N
H3C
=O PO
3
X
N
O
H
10.81
H
3H
-14CO2
H N
H3C
H2O
O
H
X
O
N
O
H
10.80
14
3H
O
PMP and [Fe-S] Cluster
E1 enzyme
The first step in the deoxygenation of CDP-4-keto-6deoxy-D-glucose (10.82) to CDP-4-keto-3,6-dideoxyD-glucose (10.83) by CDP-6-deoxy-L-threo-D-glycero4-hexulose 3-dehydratase
Me
OH
O
4
3
Me
O
OCDP
OH
10.82
PMP
O
O
OCDP
OH
10.83
coupled to E3 (see Chapter 3)
Scheme 10.48
Syn-Elimination
Comparison of a PMP-dependent elimination reaction
(A) with the corresponding tautomerization reaction (B)
pro-S
PMP in elimination
PMP
O
4'
N
+
+
H
=O
3PO
PMP in tautomerization
B:
O-
PMP
O
Scheme 10.49
H
4'
=O PO
3
B
H
O-
R
H
N
COO-
3PO
B
+
HN
R
HN
H
=O
R
-HX
X
H
O-
H
+
B:
H
R
N
X
B
H
+
HN
B
B
O- H
R
A
B:
+
B:
H
H
N
+
COO-
H R
HN
+
H
H
=O
3PO
Reaction run in H218O gives substrate with 18O in ketone
When X = OH, it is exchanged with 18OH (reversible)
COO-
Mechanism for E1
Proposed mechanism for the dehydration
catalyzed by CDP-6-deoxy-L-threo-Dglycero-4-hexulose 3-dehydratase (E1)
B
Me
B:
Me
O
O 1
OH
2
4
OCDP
OH
10.82
3
O-
PMP
-H2O
E1
HS
H
N
+
HN
H
Me H
OH
O
=O PO
3
10.84
O
N
+
+
OCDP
OH
HN
+
HR
-H2O
O-
OCDP
OH
=O PO
3
10.85
H218O
Me
Scheme 10.50
O
18O
OCDP
OH
A Syn Elimination Reaction Catalyzed by
a Catalytic Antibody Compared to the
Reaction in Solution
O
O
catalytic
antibody
F
Ph
Ph
H
CH3
H
Ph
Ph
CH3
Ph
H
FH
F
in
solution
H
CH3
Ph
Ph
H
H
staggered
Scheme 10.51
Ph
Ph
CH3
O
H
CH3
O
eclipsed
O
Ph