The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 10 Eliminations and Additions

1
The Organic Chemistry of Enzyme-Catalyzed
Reactions Chapter 10 Eliminations and
Additions
2
Reactions catalyzed by dehydratases and hydratases
Anti Eliminations and Additions
Scheme 10.1
3
Three General Mechanisms for Dehydration
(nonenzymatic)
Scheme 10.2
4
Enzymatic Dehydrations
When H is next to COOH, anti-dehydration When H
is next to aldehyde, ketone, or thioester,
syn-dehydration
5
Reaction catalyzed by enolase
M2-dependent Dehydration
Scheme 10.3
Kd 1
5.15 ppm in NMR
5.33 ppm in NMR
2-phospho-D-glyceric acid (2-PGA)
phosphoenolpyruvate (PEP)
2 Mg2 required
6
Anti- versus Syn-Elimination of Water from
2-Phosphoglycerate (PGA)
NMR 5.14 ppm therefore anti-elimination
Scheme 10.4
7
Stereochemistry of Water Addition to
Phosphoenolpyruvate Catalyzed by Enolase
Back reaction
proton adds to si-face of PEP therefore OH must
add to re-face
H2O
-H2O
PGA
Scheme 10.5
8
Relative Rates of Exchange in the
Enolase-catalyzed Reaction
slow step - release of PEP
fast step - deprotonation
therefore E1cB
9
Evidence for E1cB Mechanism for Enolase
3H exchanges into 2-PGA
2-PGA
aci-carboxylate
Scheme 10.6
10
Evidence for Aci-carboxylate Intermediate (10.3)
All are potent inhibitors
11
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 coordination lowers pKa of the C-2 H
Lys-345 is in a hydrophobic region - lowers pKa,
increases free base form
Figure 10.1
12
Reactions catalyzed by nucleoside
diphosphohexose-4,6-dehydratases
(oxido-reductases). NDP stands for nucleoside
diphosphate. The sugar positions are numbered.
NAD-Dependent Elimination
reduced
oxidized
Scheme 10.8

• dTDP-4-3H10.11 dTDP-6-3H10.14
  (intramolecular)

• In 2H2O product incorporates 2H at C-5

• All 3H released from dTDP-5-3Hglucose

• With 4-3HNAD no 3H in product (suggests
  intramolecular)

13
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 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
14
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
anti-elimination of H2O
washed out
Scheme 10.9
suprafacial 3H transfer
anti-addition of H- and H
15
Transfer of the C-4 hydrogen of (6S)-10.15 and
(6R)-CDP-4-2H, 6-3HD-glucose (10.17) to the C-6
methyl group in the CDP-4-keto-6-deoxyglucose
product
Determination of the Stereochemistry of Me Groups
chirally tritiated
chiral Me group
epimeric Me group
chirally tritiated
Scheme 10.10
16
Kuhn-Roth Oxidation of CDP-4-keto-6-deoxyglucose
S
R
Polarimetry will not work 3H only in trace
amount
Scheme 10.11
17
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
S
free rotation
Scheme 10.12
anti-elim.
(KIE 3.8)
With 10.16 71 T in fumarate therefore
(R)-acetate With 10.18 30 T in fumarate
therefore (S)-acetate
supports inversion of stereochemistry
18
Outcomes of the Malate Synthase/Glyoxylate
Reaction Followed by Hydrolysis
No 3H not detected
Slide not in text--after Scheme 10.12
19
Aconitase-catalyzed interconversion of citrate
(10.23) and isocitrate (10.25) via cis-aconitate
Iron-sulfur Clusters in a Nonredox Role
dehydration
hydration
cis-aconitate
isocitrate (2R, 3S)
citrate
Scheme 10.13
20
Citrate is Prochiral
removed in going to cis-aconitate
21
Stereochemistry of Elimination of Water from
Citrate Catalyzed by Aconitase
Scheme 10.14
must be anti-elimination to give cis-aconitate
22
Stereochemistry of Addition of Water to
Cis-aconitate to Give Citrate (back reaction)
(re-si)
anti-addition
(si-re)
Scheme 10.15
23
Stereochemistry of Addition of Water to
Cis-aconitate to Isocitrate
(re-si)
anti-addition
(si-re)
cis-aconitate
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!
24
Overall Stereochemistry of the Aconitase-catalyzed
Reaction
(si-re)
(re-si)
Scheme 10.17
25
A Crossover Experiment with Aconitase in Which
(2R)-3Hcitrate and 2-Methyl-cis-aconitate
(10.27) Produce Unlabeled Cis-aconitate and
2-Methyl-3-3Hisocitrate (10.28)
crossover product observed
Scheme 10.18
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!
26
A Proposed Mechanism for Aconitase
(after release from active site)
Scheme 10.19
27
Where does the iron-sulfur cluster come in?
from crystal structure
Fe acts as a Lewis acid - nonredox role
28
Support for Aci-carboxylate Bound to Fe-S Cluster
very potent inhibitor
very acidic
product mimic
29
Crystal structure with 10.31 bound is same as
with isocitrate bound (to Fe-S cluster) Therefore,
ElcB (carbanion) mechanism
30
Reaction catalyzed by chorismate synthase
Elimination of Phosphate
anti-elimination
EPSP
chorismate
Scheme 10.22
Orbital symmetry rules concerted 1,4-elimination
is syn - suggests stepwise elimination
31
not a substrate
Therefore not 1,3-rearrangement of phosphate
32
E1 (pathway a) and addition/elimination (pathway
b) mechanisms for chorismate synthase
Other Possibilities
E1
covalent
Scheme 10.23
33
To Test These Mechanisms
F or H
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
34
Radical Mechanism Proposed for Chorismate Synthase
Scheme 10.24
35
Chemical Model in Support of the Radical
Mechanism for Chorismate Synthase
Scheme 10.25
36
Reaction catalyzed by histidine ammonia-lyase
(HAL)
Elimination of Ammonia Ammonia Lyases
urocanic acid
dehydroalanyl-dependent
Scheme 10.26
37
Reactions to identify the active-site prosthetic
group as a dehydroalanyl moiety
Evidence for Dehydroalanyl Enzyme
Ala
Asp
Scheme 10.27
Dbu
38
Posttranslational conversion of the active site
Ala-Ser-Gly at positions 142-144 to give a
dehydroalanyl-like species
Actual Prosthetic Group is Not Dehydroalanyl, but
Something Related
Ala-Ser-Gly
crystal structure
Scheme 10.28
39
Stereochemistry of the Elimination Catalyzed by
Histidine Ammonia-lyase
Scheme 10.29
In 3H2O 3-pro-R hydrogen of His is exchanged
(lost in conversion to urocanate)
(anti-elimination)
(reversible)
His 2-14Curocanate ? 14CHis
(reversible)
urocanate NH3 ? His
40
Initial Proposed Mechanism for Histidine
Ammonia-lyase
Scheme 10.30
Whats wrong with this mechanism?
The pKa of the proton being abstracted is very
high.
41
Activation of C-3 Deprotonation by a 2?-Nitro
Group in the Histidine Ammonia-lyase Reaction
Scheme 10.31
This is a good substrate even with mutants that
do not contain dehydroalanyl-like group
42
Proposed alternative (electrophilic aromatic
substitution) mechanism for histidine
ammonia-lyase
Alternative Role for the Prosthetic Group
makes the C-3 proton more acidic
electrophilic aromatic substitution
Scheme 10.32
43
Reaction catalyzed by 3-dehydroquinate
dehydratase (3-dehydroquinase)
Syn-Eliminations and Additions
Scheme 10.33
syn-elimination
3-dehydroquinate
3-dehydroshikimate
NaBH4 inactivates the enzyme in the presence of
substrate One 3H incorporated into protein with
NaB3H4 substrate
44
Proposed mechanism for 3-dehydroquinate
dehydratase (3-hydroquinase)
Schiff Base Mechanism
detected by electrospray MS
ElcB
Scheme 10.34
45
Pyridoxal 5?-phosphate-dependent ?-elimination
(A) and ?-elimination (B) reactions
PLP-dependent Eliminations
?-elimination
?-elimination
Scheme 10.35
46
Pyridoxal 5?-Phosphate-dependent ?-Replacement
(A) and ?-Replacement (B) Reactions
?-replacement
?-replacement
Scheme 10.36
47
Proposed Mechanism for PLP-dependent
?-Elimination Reactions
detected spectrally
detected spectrally
detected by NaBH4 treatment
Scheme 10.37
48
Proposed Mechanism for PLP-dependent
?-Replacement Reactions
Scheme 10.38
49
Reaction Catalyzed by Tryptophan Synthase
Scheme 10.39
?2?2 tetramer ? subunits contain PLP - catalyze
?-elimination part ? subunits needed for ?
replacement
50
Proposed Mechanism for Tryptophan Synthase in the
Absence and Presence of ? Subunits
Scheme 10.40
detected by NMR
comes from ?-H
same result in D2O (still get H incorporated)
With
R
Suprafacial syn-elimination from si face
51
Proposed Mechanism for the Reaction Catalyzed by
Tryptophanase
This is a leaving group
transferred from C-2
Scheme 10.41
This is not a leaving group
syn-elimination
suprafacial 1,3H transfer
detected
KIE 3.6
Exact reverse of Trp synthase
retention of configuration
52
Stereochemical Differences between Trp Synthase
and Tryptophanase
Have opposite inhibitory potencies with the two
enzymes therefore opposite stereochemistry
53
Proposed Difference in the Stereochemistry of the
Reactions Catalyzed by Tryptophanase and
Tryptophan Synthase
Scheme 10.42
54
Reactions catalyzed by ?-cystathionase (A) and
cystathionine ?-synthase (B)
?-Elimination and ?-Replacement
Scheme 10.43
?-cystathionase
(?-elimination)
cystathionine
(?-replacement)
cystathionine ?-synthase
O-succinyl-L-homoserine
Some internal return (not 100) therefore
suprafacial
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Proposed Mechanism for the Reaction Catalyzed by
PLP-dependent ?-Elimination Enzymes
pro-R
only partial internal return
solvent H
Scheme 10.44
Hx represents solvent protons. HxHbHaB implies
that one or more of these protons is attached to
the base B.
56
Proposed Mechanism for the Reaction Catalyzed by
PLP-dependent ?-Replacement Enzymes.
pro-R
syn-elimination
solvent H (suprafacial)
Hx represents solvent protons. HxHbHaB implies
that one or more of these protons is attached to
the base B.
Scheme 10.45
57
Mechanism-based Inactivator of ?-Cystathionase
Incorporates 2 mol radioactivity/mol tetrameric
enzyme (half-sites reactivity) with covalent
attachment to enzyme ?-2H10.74 KIE 2.2 on
inactivation Demonstrates removal of C-2 proton
for inactivation
58
Acid hydrolysis of radiolabeled enzyme gives
59
Mechanism-based Inactivation of ?-Cystathionase
by Propargylglycine
inactivated enzyme
Scheme 10.46
60
Another Mechanism-based Inactivator of
?-Cystathionase
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
3HGly
61
Mechanism-based Inactivation of ?-Cystathionase
by ?,?,?-Trifluoroalanine
Scheme 10.47
?max 519 nm
3HGly
62
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
PMP and Fe-S Cluster E1 enzyme
coupled to E3 (see Chapter 3)
Scheme 10.48
63
Comparison of a PMP-dependent elimination
reaction (A) with the corresponding
tautomerization reaction (B)
Syn-Elimination
pro-S
PMP in elimination
PMP in tautomerization
Scheme 10.49
Reaction run in H218O gives substrate with 18O in
ketone When X OH, it is exchanged with 18OH
(reversible)
64
Proposed mechanism for the dehydration catalyzed
by CDP-6-deoxy-L-threo-D-glycero-4-hexulose
3-dehydratase (E1)
Mechanism for E1
Scheme 10.50
65
A Syn Elimination Reaction Catalyzed by a
Catalytic Antibody Compared to the Reaction in
Solution
Scheme 10.51