the organic chemistry of enzyme-catalyzed reactions chapter 10 eliminations and additions
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The Organic Chemistry of Enzyme-Catalyzed Reactions
Chapter 10
Eliminations and Additions
Anti Eliminations and Additions
Scheme 10.1
Reactions catalyzed by dehydratases and hydratases
R C
H
R'
C R''
H
OH
C CHR''R
R'+H2O
-H2O
Scheme 10.2
Three General Mechanisms for Dehydration (nonenzymatic)
C
C OH
H
C
C OH
H C
C OH
C
CHO
H C
C
carbocationmechanism
slow
fast
stabilized carbanion
C
C
carbanionmechanism
HB
+ OHE1cB
C
C OH2
H
E2+ H2O
C
C
H
concertedmechanism
E1
fast
C
C
slow
B
B
B
Enzymatic Dehydrations
When H is next to COOH, anti-dehydration
When H is next to aldehyde, ketone, or thioester, syn-dehydration
Scheme 10.3
M2+-dependent Dehydration
Kd ~ 1
2 Mg2+ required
2-phospho-D-glyceric acid
(2-PGA)
5.33 ppm in NMR
5.15 ppm in NMR
phosphoenolpyruvate(PEP)
Reaction catalyzed by enolase
COO-
H
HA
OH
HB
OPO3=
HA HB
-OOC OPO3=
10.210.1
+H2O
-H2O
Scheme 10.4
NMR 5.14 ppm; therefore anti-elimination
Anti- versus Syn-Elimination of Water from 2-Phosphoglycerate (PGA)
H2H
OHOPO3
=-OOC
H
H
2H
OH
HC
2H
-OOCC
OPO3=
2HC
H
-OOCC
OPO3=
OPO3=-OOC
H
anti
syn
(3R)-[3-2H]-2-PGA
+H2O
-H2O
Scheme 10.5
Back reaction
+H2O
-H2O
proton adds to si-face of PEP; therefore OH must add to re-face
Stereochemistry of Water Addition to Phosphoenolpyruvate Catalyzed by Enolase
H OPO3=
COO-H H
OPO3=
COO-
H H
HO2R
si
re
PGA
krel
14
PEP
14
PGA 1.0[ 3-
18
O] PGA H2
18
O 1.3
[ 2-
2
H] PGA
2
H2
O 1.9
slow step - release of PEP
fast step - deprotonation
Relative Rates of Exchange in the Enolase-catalyzed Reaction
therefore E1cB
Scheme 10.6
3H exchanges into 2-PGA
aci-carboxylate
Evidence for E1cB Mechanism for Enolase
C
C
C
OH
OPO3=
H
H
H
B:
B
C
CH2
-OOC OPO3=
B
B+H
C
C
C OPO3=
fast
slowH2O +
H
OH
O
O
H
O O-
3H2O
10.3
M2+
3H
HM2+
2-PGA
All are potent inhibitors
Evidence for Aci-carboxylate Intermediate (10.3)
NPO3
=
HO
O
O PO3=
HO
O
ON
PO3=
O
OPO3
=
NHHO
O
10.4 10.5 10.6 10.7
C
C
C OPO3=
H
OH
O
O
H
10.3
M2+
Figure 10.1
Mg2+ coordination lowers pKa of the C-2 H+ Lys-345 is in a hydrophobic region - lowers pKa, increases free base form
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+
CC
O
O
Mg2+
O
HCH2
HOHO
O
211Glu
P
O O
O
NH2 Lys345
Mg2+
Scheme 10.8
NAD+-Dependent Elimination
oxidized reduced
• dTDP-[4-3H]10.11 dTDP-[6-3H]10.14 (intramolecular)
Reactions catalyzed by nucleoside diphosphohexose-4,6-dehydratases (oxido-reductases). NDP stands for
nucleoside diphosphate. The sugar positions are numbered.
10.11
OHO
OH
HOHO
ONDP
OOH
HOHO
ONDP
O
O
OHONDP
HO
O OCH3
HOHO
ONDP10.1310.12
O
10.14
123
4 56
NAD+ NAD+NADHNADH
• With [4-3H]NAD+ no 3H in product (suggests intramolecular)
• All 3H released from dTDP-[5-3H]glucose
• In 2H2O product incorporates 2H at C-5
Test for Intramolecular or Intermolecular H Transfer
Crossover experiment: labeled and unlabeled substrates added together and look for transfer of atom or group to other substrateIf 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
10.11
OHO
OH
HOHO
ONDP
OOH
HOHO
ONDP
O
O
OHONDP
HO
O OCH3
HOHO
ONDP10.1310.12
O
10.14
123
4 56
NAD+ NAD+NADHNADH
C-4 transferred to C-6 intramolecularly
OO
OD
DOOD
ONDP
3H
NR
HD2NO
D
B:
O
OD
DOOD
ONDP
NR
H
D2NO
B
3H
D
O
H
BD
B:
O
DOOD
ONDP
NR
H
D2NO
B
3H
D
O
O
OD
DOOD
ONDP
NR
H
D2NO
B:
3H
O
BD
BH
D
BD
O
DOOD
ONDP
B:
O
BD
NR
HD2NO
DO
DOOD
ONDP
O
D
NR
HD2NO
BD
10.11
B:
10.12
10.1310.14
3H
H H3H
H H
D2O
Scheme 10.9
anti-elimination of H2O
anti-addition of H- and H+
washed out
suprafacial 3H transfer
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
OHO
HO
HOOH
OCDP
2H H
3H
OHO
HO
HOOH
OCDP
2H 3H
H
10.15
10.17
CDP-D-glucose 4,6-dehydratase OCH2H3H
HOOH
OCDP
O
CDP-D-glucose 4,6-dehydratase OCH3H2H
HOOH
OCDP
O
10.16
10.18Scheme 10.10
Determination of the Stereochemistry of Me Groups
chirally tritiated chiral Me group
epimeric Me group
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
Scheme 10.11
S R
Polarimetry will not work; 3H only in trace amount
Kuhn-Roth Oxidation of CDP-4-keto-6-deoxyglucose
10.16
OCH2H3H
HOOH
OCDP
O 30% aq. CrO3
H2SO4155 oC
orCOOH
H
TD
COOH
H
DT
H
COOH
T
DSCoA
O
H
T
D
COOHT
D
HOOC H
OH
COOH
H
T
HOOC H
OH
COOHD
2. phosphotransacetylase/CoASH
HOOC H
1. malate synthase/glyoxylate
2. hydrolysis
COOHT
fumarase
10.19
HOOC H
80%
+
fumarase
1. acetate kinase/ATP+
10.20
20%10.21 10.22
-3H2O -H2O
Scheme 10.12
get both products(only detecting 3H products)
2S-malate 2S
anti-elim.
free rotation
S
With 10.16: 71% T in fumarate; therefore (R)-acetate
With 10.18: 30% T in fumarate; therefore (S)-acetate
Enzymatic Conversion of Chiral Methyl-containing Acetate into Fumarate for Determination of the Chirality of the Methyl Group
supports inversion of stereochemistry
HOOC
OH
H
COOH
T
D
HOOC
OH
H
COOH
H
T
10.19 10.20 (KIE = 3.8)
Outcomes of the Malate Synthase/Glyoxylate Reaction Followed by Hydrolysis
SCoA
O
H
T
D
COSCoAT
D
HOOC H
OH
COOH
H
T
HOOC H
OH
10.20
B:
SCoA
OT
D
HOOC
O
H
H B
hydrolysis
COOHT
D
HOOC H
OH
10.19
SCoA
O
D
H
T
COSCoAH
T
HOOC H
OH
B:
SCoA
OH
T
HOOC
O
H
H B
hydrolysis
COOH
D
H
HOOC H
OH
SCoA
O
T
D
H
COSCoAD
H
HOOC H
OH
B:
SCoA
OD
H
HOOC
O
H
H B
hydrolysis
Slide not in text--after Scheme 10.12
No 3H; not detected
COO-
COO-
H
-OOC
H
H
OH
H
-OOC
CH2
COO-
COO-
-OOC
H
COO-
H
-OOC
H
OH
H
H
10.2310.24
10.25
-H2O
+H2O -H2O
+H2O
Scheme 10.13
Iron-sulfur Clusters in a Nonredox Role
citrate
dehydration hydration
cis-aconitate isocitrate (2R, 3S)
Aconitase-catalyzed interconversion of citrate (10.23) and isocitrate (10.25) via cis-aconitate
removed in going to cis-aconitate
Citrate is Prochiral
COO-
COO-
HS
-OOC
HR
HR
OH
HS
pro-R arm
pro-S arm
10.26
Scheme 10.14
must be anti-elimination to give cis-aconitate
Stereochemistry of Elimination of Water from Citrate Catalyzed by Aconitase
-OOC
OH-OOC HS
HRCOO-
H-OOC
-OOC
COO-
B:
H B
2
3
10.2310.24
Scheme 10.15
(re-si)
anti-addition
(si-re)
Stereochemistry of Addition of Water to Cis-aconitate to Give Citrate (back reaction)
-OOC
OH-OOC H
H COO-
COO-
-OOC H
COO-
3
2re
si
10.2310.24
H
HO-
H2O
-OOC
H-OOC H
HO COO-
COO-
-OOC
H
COO-
10.2510.24
3 2
resi
H+
-OH
H2O
Scheme 10.16
(re-si)
(si-re)
anti-addition
cis-aconitate isocitrate (2R, 3S)
Therefore C-2 is always attacked on the face opposite attack at C-3
Stereochemistry of Addition of Water to Cis-aconitate to Isocitrate
Labeling studies show that the pro-R proton removed from C-2 of citrate ends up at C-3 of isocitrate!
Scheme 10.17
(si-re) (re-si)
Overall Stereochemistry of the Aconitase-catalyzed Reaction
COO-
-OOC
H COO-
COO-
-OOC H
COO- *H+
isocitratecitrateflip
*H+
HO-HO-
-H2OH2O
Scheme 10.18crossover product
observed
Therefore the proton removed from one substrate molecule can be transferred to a different substrate molecule (intermolecular)
A Crossover Experiment with Aconitase in Which [(2R)-3H]citrate and 2-Methyl-cis-aconitate (10.27) Produce Unlabeled Cis-aconitate and 2-Methyl-[3-
3H]isocitrate (10.28)COO-
CH 3H
C
CH2
OH-OOC
COO-
-OOC CH3
-OOC
COO-
-OOC H
-OOC
COO-
COO-
CH3C OH
C
CH2
3H-OOC
COO-
+ aconitase+
10.27 10.28
The OH exchanges with solvent, but the proton removed does not!
Scheme 10.19
A Proposed Mechanism for Aconitase
X
B:H
COO-
COO-
-OOC
3H
H COO-
18OH
COO-
X
B
X
B:
X
B
COO-
HCOO-
-OOC
3H
COO-
OH
COO-H
-OOC
3H++
flip18
3H18
-OOC
OH
OH
OH
OH
(after releasefrom active site)
from crystal structure
Fe acts as a Lewis acid - nonredox role
Where does the iron-sulfur cluster come in?
Fe
S Fe
Fe S -O
OH
OC
O
H
OH
165Asp
O
COO-
COO-
H
10.29
SFe
S
protein
protein
protein
H
very potent inhibitor
product mimic
Support for Aci-carboxylate Bound to Fe-S Cluster
COO-
COO-
H
C
H
OH
H
H
COO-
COO-
H
N
H
OH
H
H
O
O
O
O
isocitrate10.30
very acidic
Crystal structure with 10.31 bound is same as with isocitrate bound (to Fe-S cluster)Therefore, ElcB (carbanion) mechanism
COO-
COO-
H
C
H
OH
H
COO-
COO-
H
N
H
OH
H
O
O
O
O
10.3210.31
COO-
OH
=O3PO OCOO-
HR
HS
COO-
OH
OCOO-
+ PO43- + HR
+
10.4010.39
12
34
5
6
HS
Scheme 10.22
Elimination of Phosphate
anti-elimination
EPSP chorismate
Orbital symmetry rules: concerted 1,4-elimination is syn - suggests stepwise elimination
Reaction catalyzed by chorismate synthase
Therefore not [1,3]-rearrangement of phosphate
not a substrate
COO-
OH
OCOO-
10.41
=O3PO
COO-
OH
OCOO-
10.41
=O3POCOO-
OH
OCOO-
=O3PO
H
COO-
OH
OCOO-
:B
Scheme 10.23
Other Possibilities
E1
covalent
E1 (pathway a) and addition/elimination (pathway b) mechanisms for chorismate synthase
COO-
OH
=O3PO OCOO-
H
H
COO-
OH
OCOO-
- X
COO-
OH
OCOO-
H
H
:B
COO-
OH
X
OCOO-
H
H
:B
a
COO-
OH
-X
OCOO-
H
H
- PO43-
a
b
+
b
- PO43-
E2
c
E1
:B
d
F or H
Neither was a substrate nor an inactivator
To Test These Mechanisms
COO-
OH
=O3PO OCOO-
RR
RS
10.42
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
Scheme 10.24
Radical Mechanism Proposed for Chorismate Synthase
H
O
OH
=O3PO
CO2-
CO2- O
OH
=O3PO
CO2-
CO2-
O
OH
CO2-
CO2-
O
OH
CO2-
CO2-
O
OH
CO2-
CO2-
+ e--H
Scheme 10.25
Chemical Model in Support of the Radical Mechanism for Chorismate Synthase
CO2Me
OTBDMS
P
Br
CO2Me
OTBDMS
O
(PhO)2 O
CO2Me
OTBDMS
P
O
(PhO)2 O
-(PhO)2PO2SnBu3
Scheme 10.26
Elimination of Ammonia: Ammonia Lyases
urocanic acid
dehydroalanyl-dependent
Reaction catalyzed by histidine ammonia-lyase (HAL)
N
NH
COO-
NH3+
D
D D
N
NH
COOH
H
D
[D3]His 10.43
HAL
5'
12
3H2O
Scheme 10.27
Evidence for Dehydroalanyl Enzyme
Ala
Asp
Dbu
Reactions to identify the active-site prosthetic group as a dehydroalanyl moiety
NH
NH
O
3HH2CNH
NH
O3HH2C COOH
NH3+
NH
NH
O
N14CCOOH
NH3+
HOO14C
14CH2 O
+NH3
O2N
NH
NHNH3
+
COO-
10.45
1. H2/Pt2. 6 N HCl 14
10.44
NaB3H4H3O+
Δ
H3O+
Δ
14CH2NO2
Δ
14CN
Scheme 10.28
Actual Prosthetic Group is Not Dehydroalanyl, but Something Related
Ala-Ser-Glycrystal structure
Posttranslational conversion of the active site Ala-Ser-Gly at positions 142-144 to give a
dehydroalanyl-like species
B H B
NH
HN
NH
HN
OOH
O
O N
N
H OH
NH
O
O
HN
N
NNH
O
O
HN
..
10.45a
143144142
Scheme 10.29
His + [2-14C]urocanate [14C]His
(anti-elimination)
Stereochemistry of the Elimination Catalyzed by Histidine Ammonia-lyase
N
NH
NH3+
COOH
H
HS 3HR
N
NH
COOH
H
-3HR+
-NH3
urocanate + NH3 His
(reversible)
(reversible)
In 3H2O 3-[pro-R] hydrogen of His is exchanged (lost in conversion to urocanate)
Scheme 10.30
What’s wrong with this mechanism?
Initial Proposed Mechanism for Histidine Ammonia-lyase
NH
NH
O
COO-
NH2
N
NH
NH
NH
O
COO-
NH2
N
NH
BH H
:B
H B
NH
NH
O
COO-
NH2
N
NH
:BH H
NH
NH
O
COO-
NH2
N
NH
NH
NH
O
COO-
NH2
N
NH
D
NH
NH
O
COO-
NH3
N
NH
HB
NH
NH
O
D2O
-NH3
The pKa of the proton being abstracted is very high.
Scheme 10.31
This is a good substrate even with mutants that do not contain dehydroalanyl-like group
Activation of C-3 Deprotonation by a 2-Nitro Group in the Histidine Ammonia-lyase Reaction
COO-
NH2N NH
N
O
OHH
COO-
NH2N NH
N
O
OB
COO-
NH3N NH
HH
BH
COO-
NH3N NH
HH
:B
COO-
NH3N NH
H
COO-
N NH
H
:B
COO-
N NH
:B
:B
10.46 10.47
N
NNH
O
10.45a
N
NNH
OH
N
NNH
OH
N
NNH
OH
10.48
BH
N
NNH
O
10.45a
:
:
-NH3
Scheme 10.32
Alternative Role for the Prosthetic Group
makes the C-3 proton more acidic
electrophilic aromatic substitution
Proposed alternative (electrophilic aromatic substitution) mechanism for histidine ammonia-lyase
Scheme 10.33
Syn-Eliminations and Additions
syn-elimination
3-dehydroquinate 3-dehydroshikimate
NaBH4 inactivates the enzyme in the presence of substrateOne 3H incorporated into protein with NaB3H4 + substrate
Reaction catalyzed by 3-dehydroquinate dehydratase (3-dehydroquinase)
H
COO-
OH
OH
O
HS
HRHO COO-
OH
OH
O
10.49 10.50
+ HR
Scheme 10.34
Schiff Base Mechanism
detected by electrospray MS
ElcB
Proposed mechanism for 3-dehydroquinate dehydratase (3-hydroquinase)
HS
HR
HO COO-
OH
OH
O
HS
HRHO COO-
OH
OH
HN
HS
HO COO-
OH
OH
HN
BH
Lys
COO-
OH
OH
HN
Lys
COO-
OH
OH
HN
Lys
Lys
LysNH2
COO-
OH
OH
O++
Lys
NH2
NaBH4
H2O
B:
Scheme 10.35
PLP-dependent Eliminations
-elimination
-elimination
Pyridoxal 5-phosphate-dependent -elimination (A) and -elimination (B) reactions
R CH
X
CHCOO-
NH3+
RCH C
NH3+
COO- RCH2 C
O
COO-
CH2 CH2 CHCOO-
NH3+
CH3CH C
NH3+
COO- CH3CH2 C
O
+ NH4+E•PLP
E•PLP + NH4+COO-X
H2OA
H2OB
Scheme 10.36
-replacement
-replacement
Pyridoxal 5-Phosphate-dependent -Replacement (A) and -Replacement (B) Reactions
R CH
X
CHCOO-
NH3+
CH2 CH2 CHCOO-
NH3+
E•PLP
E•PLPX
R CH
Y
CHCOO-
NH3+
CH2 CH2 CHCOO-
NH3+
Y
AY-
BY-
NN
H
OH3C H
=O3PO
DX
H3H
COO-
:B
NH3+
DX
COO-
H
3H HB
NN
H
OH3C H
=O3PO
X
H3H
COO-
B
B
D
H
NN
H
OH3C H
=O3POH
3H
COO-
B
B
DB
B-OOC
NH
D
3HH
NN
H
OH3C
H
=O3PO H3H
COO-
B
BD
+ E•PLP
+ E•PLP
-OOC
O
D
3HH
10.51 10.52 10.53
NH2
10.54
NN
H
OH3C
H
=O3PO H3H
COO-
NH
D
+ E•PLP
b
aa
b
-OOC
O
D
3HH
NH3 +
+ NH3
10.55
10.56
10.5710.56
..
::
H2O
H2O
-XH
Scheme 10.37
detected spectrallydetected spectrally
detected by NaBH4 treatment
Proposed Mechanism for PLP-dependent -Elimination Reactions
Scheme 10.38
Proposed Mechanism for PLP-dependent -Replacement Reactions
NN
H
OH3C H
=O3PO
DX
H3H
COO-
:B
NH3+
DX
COO-
H
3H
E•PLP +
+ E•PLP
HB
NN
H
OH3C H
=O3PO
X
H3H
COO-
B
B
D
H
NN
H
OH3C H
=O3POH
3H
COO-
B
D
NN
H
OH3C H
=O3PO
Y
H3H
COO-
BD
NN
H
OH3C H
=O3PO
DY
H3H
COO-
:B
NH3+
DY
COO-
H
3H
10.51 10.52 10.53
10.54
Y
-XH
Scheme 10.39
22 tetramer
subunits contain PLP - catalyze -elimination part
subunits needed for replacement
Reaction Catalyzed by Tryptophan Synthase
CHCOO-
NH3+
HOCH2
NH
COO-
NH3+
NH
PLP+ + H2O
2
3
indole Trp
Scheme 10.40
detected by NMR
Withsame result in D2O (still get H incorporated)
Suprafacial syn-elimination from si face
comes from -H
Proposed Mechanism for Tryptophan Synthase in the Absence and Presence of Subunits
R
HOCOO-
NH3+
HOCOO-
NH+
B:3H
NH
O-
3H
COO-
NH+
NH
O-
=O3PO
HOCOO-
NH+
NH
O-=O3PO
=O3PO
B 3H
NH
COO-
NH+
NH
O-=O3PO
NH
COO-
NH+
NH
O-=O3PO
B: B+
H
NH
COO-
NH+
NH
O-=O3PO
10.58
NH
COO-
NH+
B:H
NH
O-=O3PO
+
E•PLP
10.58
+
E•PLP + Trp
O
COO-in the absence
or indole
pyruvate
+ NH4+ + E•PLP
in the presence
and indole
..
..
..
..
of a subunits
of subunits
B H
-H2O
B 3H3H
H
H2O
HO
3H2H
COO-
NH3+
H
Trp
synthase
H
3H2H
COO-
O
NH
COO-
NH3+
NH
CH3C
O
COO-
NH
COO-
NH+
B:3H
NH
O-
NH
COO-
NH+
B+3H
NH
O-
3H
3H
COO-
NH+
NH
O-
NH2
=O3PO=O3PO
NH
COO-
NH+
NH
O-
+
10.59
=O3PO
10.60
10.61
3H
E•PLP
=O3PO
COO-
NH
NH
O-=O3PO
NH2COO-
NH2
+
B H
+ NH4+
10.58
COO-
NH2
..
..H3C
:
E•PLP
H2O
Scheme 10.41
This is not a leaving group
This is a leaving group
transferred from C-2
syn-eliminationsuprafacial [1,3]H+ transfer
detected
Exact reverse of Trp synthaseretention of configuration
Proposed Mechanism for the Reaction Catalyzed by Tryptophanase
2H
H3H
COO-
O
2H2O
H3H
COO-
NH3+
H
NH KIE 3.6
Have opposite inhibitory potencies with the two enzymes; therefore opposite stereochemistry
Stereochemical Differences between Trp Synthase and Tryptophanase
NH
COO-
NH3+
* *
RS
SS
10.62
Scheme 10.42
Proposed Difference in the Stereochemistry of the Reactions Catalyzed by Tryptophanase and Tryptophan Synthase
N
NH
COO-
NH+
NH
O-=O3PO
H
NH
COO-
NH+
NH
O-
re,re
si,si=O3PO
tryptophanase
tryptophan synthase
NH
COO-
NH+
NH
O-=O3PO
H
H
Scheme 10.43
-Elimination and -Replacement
-cystathionase(-elimination)
(-replacement)cystathionine
O-succinyl-L-homoserine cystathionine -synthase
Some internal return (not 100%); therefore suprafacial
Reactions catalyzed by -cystathionase (A) and cystathionine -synthase (B)
NH3+
H
COO-S
NH3+
H-OOC
SH
NH3+
H-OOC O
COO-
NH3+
H
COO-S
NH3+
H-OOCNH3
+
H
COO-O
O
-OOC
10.63
SH
E•PLP
NH3+
H-OOC
+
+E•PLP
10.64
+ NH3
succinic acid
B
A
Scheme 10.44
pro-R
Proposed Mechanism for the Reaction Catalyzed by PLP-dependent -Elimination Enzymes
NN
H
OH3C H
=O3PO
HaHb
Hc
COO-
HxB
NH3+
Ha
COO-N
NH
OH3C H
=O3PO
Hb
Hc
COO-
HxHaB
NN
H
OH3C H
=O3PO
Hc
COO-
HxHaHbB
NN
H
OH3C H
=O3POHc
COO-
+ E•PLPX
Hb
He
Hc
Hd
10.67
X
HeHd
+ E•PLP+ NH3
X
HdHe
X
HdHe
..HxHaHbB
HdHe
NN
H
OH3C H
=O3POHc
COO-
..Ha,b,x
He Hd
HxHaHbB
NN
H
OH3C H
=O3PO Hc
COO-
Ha,b,x
He Hd
Hx
Hc
COO-
10.65
Ha,b,x
He Hd
10.69
10.70
Hx
10.66
O
10.68
..
H2O
Hx represents solvent protons. HxHbHaB implies that one or more of these protons is attached to the base B.
solvent H+
only partial internal return
Scheme 10.45
solvent H+ (suprafacial)
pro-R
syn-elimination
Proposed Mechanism for the Reaction Catalyzed by PLP-dependent -Replacement Enzymes.
NN
H
OH3C H
=O3PO
HaHb
Hc
COO-
HxB
NH3+
Ha
COO-N
NH
OH3C H
=O3PO
Hb
Hc
COO-
HxHaB
NN
H
OH3C H
=O3POHc
COO-HxHaHbBN
NH
OH3C H
=O3PO
Hc
COO-
XHb
He
Hc
Hd
X
HeHd
X
HdHe
X
HdHe
HxHaHbB
HdHe
NN
H
OH3C H
=O3PO Hc
COO-
Y
He Hd
Hx
NN
H
OH3C H
=O3POHc
COO-
..
Y
HdHe
HxHaHbB
HxHaHbB
NN
H
OH3C H
=O3PO Hc
10.72
10.71
10.65
+ E•PLP
10.67
Y
He Hd
Hx
10.66
COO-
Hx
+ E•PLP
H3N
HcY
He Hd
Hx
10.68
COO-
Hx10.73
..
......
Y
Hx represents solvent protons. HxHbHaB implies that one or more of these protons is attached to the base B.
Incorporates 2 mol radioactivity/mol tetrameric enzyme(half-sites reactivity) with covalent attachment to enzyme[-2H]10.74 KIE 2.2 on inactivationDemonstrates removal of C-2 proton for inactivation
Mechanism-based Inactivator of -Cystathionase
COO-
NH3+
14
10.74
Acid hydrolysis of radiolabeled enzyme gives
COO-
NH3+
14
O
10.75
Scheme 10.46inactivated enzyme
Mechanism-based Inactivation of -Cystathionase by Propargylglycine
NN
H
OH3C H
=O3PO
HH
H
COO-
HxB
NN
H
OH3C H
=O3PO
H
H
COO-
HxHB
NN
H
OH3C H
=O3POH
C
COO-
HxHHB
NN
H
OH3C H
=O3PO
H
COO-HxHHB
C
COO-
NH3+
HC
XHH2C
X
NN
H
OH3C H
=O3PO
H
COO-HxHHB
CH2C X
10.77
NN
H
OH3C H
=O3PO H
COO-
HxHHB
C
10.76
H2C X
Hx
+ E•PLP
NN
H
OH3C H
=O3PO HC
H2C X
HxHx
COO-
..
..
....
PLP + 10.75H3O+
O
COOHH3N
2 mol/tetramer3 F- released/mol inactivator incorporatedmax = 519 nm
Denaturation releases all radioactivity as 14CO2
Denaturation in 3H2O incorporates one 3H into
enzyme; hydrolysis gives [3H]Gly
Another Mechanism-based Inactivator of -Cystathionase
F3C COO-
NH3+
10.78
NN
H
OH3C H
=O3PO
HF
F
14COO-
HxB
NN
H
OH
H3C H
=O3PO
F
FF
14COO-
HxHB
F
F3C 14COO-
NH3+
NN
H
OH3C H
=O3POF
14COO-
F
X
NN
H
OH3C H
=O3PO
14COO-
X
NN
H
OH3C H
=O3PO
X
3H O
O
NN
H
OH3C H
=O3PO
X
FF
14COO-
NN
H
OH3C H
=O3POX
14COO-
F
NN
H
OH3C H
=O3POX
F
14COO-
NN
H
OH3C H
=O3POC
X
14COO-
O
O
NN
H
OH3C H
=O3PO
10.79
-14CO2
X
+ E•PLP
3H10.80
O
H14
H3NCOOH
10.813H
PLP +H
H2OH
..
..
....
3H3O+
-F-
-F-
-F-H2O
H3O+
Scheme 10.47
[3H]Gly
max 519 nm
Mechanism-based Inactivation of -Cystathionase by ,,-Trifluoroalanine
Scheme 10.48
PMP and [Fe-S] Cluster
E1 enzyme
coupled to E3 (see Chapter 3)
The first step in the deoxygenation of CDP-4-keto-6-deoxy-D-glucose (10.82) to CDP-4-keto-3,6-dideoxy-D-glucose (10.83) by CDP-6-deoxy-L-threo-D-glycero-
4-hexulose 3-dehydratase
Me
O OH
OHOCDP
OMe
O
OHOCDP
O
43
PMP
10.82 10.83
OCOO-
RH
H
N
H
B:
=O3PO
O-
HN COO-
R
H
N
HB
=O3PO
O-
HN COO-
R
B:H
H
O
RH
H
N
H
B:
=O3PO
O-
HN
R
X
X
H
N
H
PMP+
=O3PO
O-
HN
++
R
B
+
H
B
PMP
H
+ ++
B
H
B:
+
4'
4'
A
B
-HX
Scheme 10.49
Syn-Elimination
PMP in eliminationpro-S
PMP in tautomerization
Reaction run in H218O gives substrate with 18O in ketone
When X = OH, it is exchanged with 18OH (reversible)
Comparison of a PMP-dependent elimination reaction (A) with the corresponding tautomerization reaction (B)
Scheme 10.50
Mechanism for E1
Proposed mechanism for the dehydration catalyzed by CDP-6-deoxy-L-threo-D-glycero-4-hexulose 3-dehydratase (E1)
Me
O OH
OHOCDP
OHS
HR
N
HMe
OH
OHOCDP
O
B: Me
N
H
OHOCDP
O
=O3PO
O-
HN
10.8410.82
+
+
E1
=O3PO
O-
HN
10.85
++
PMP
B
H
1
2
34
-H2O -H2O
H218O
Me
18O
OHOCDP
O
Scheme 10.51
A Syn Elimination Reaction Catalyzed by a Catalytic Antibody Compared to the
Reaction in Solution
Ph Ph
O F
CH3H
H CH3
HF
Ph
HPh
O
H
CH3
F
CH3
HPhH
Ph
OPhH
CH3
catalytic antibody
insolution
staggered
eclipsed
Ph
O
Ph
O
Ph