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![Page 1: The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 2 Group Transfer Reactions: Hydrolysis, Amination, Phosphorylation.](https://reader035.fdocuments.us/reader035/viewer/2022081503/56649d625503460f94a44ebf/html5/thumbnails/1.jpg)
The Organic Chemistry of Enzyme-Catalyzed Reactions
Chapter 2
Group Transfer Reactions: Hydrolysis, Amination,
Phosphorylation
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Hydrolysis Reactions
Amide Hydrolysis
Peptidases (proteases if protein hydrolysis involved) catalyze the hydrolysis of peptide bonds
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Scheme 2.1
NH3 CH C
O
NH COO- NH3
R1
CH
R2
CH C
O
NH C
R3
CH
R4
NH3 CH C
O
R1 R2
C
O
NH CH
R3
C
O
NH CH
R4
C+
+ ++
O
NH
NH
O
P1' P2'
S2 S1 S1' S2'
HN CH
H2O
P1P2
Reaction catalyzed by peptidases
scissile bond
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Figure 2.1
NH3 CH C
O
NH
R1
CH
R2
C
O
NH CH
R3
C
O
NH CH
R4
COO-+
exopeptidase(carboxypeptidase)endopeptidase
exopeptidase(aminopeptidase)
Classifications of peptidases
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Endopeptidases
• Representative example is -chymotrypsin• Regiospecifically hydrolyzes peptide bonds of
the aromatic acids• P1 -chymotrypsin is Phe, Tyr, and Trp• P1 for trypsin is Arg and Lys
NH3 CH C
O
NH
R1
CH
R2
C
O
NH CH
R3
C
O
NH CH
R4
COO+
P1
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Scheme 2.2
EndopeptidaseR C
O
X
Ser195 O
H
NN H
His57
- O C
O
Asp102
C
O
XR
O
Ser
NN
H
His
H -O C
O
Asp
C
O
RO
Ser
His
H
HOH
- O C
O
NN Asp
C
OH
RO
O
Ser
NN
H
His
H -O C
O
Asp
R COOHSer195OH
NN H
His57
- O C
O
Asp102
+
++
+
acyl intermediate
+
acylation
deacylation
-XH
Mechanism for -chymotrypsin
showing catalytic triad
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Figure 2.2
Evidence for Acyl Intermediate
NO2OCH3C
O
NO2O
initial burst phase
)
A400 nm
steady state phase
corresponds to 1 equivper equiv of enzyme
Time
-
(Release of
2.1
Reaction of chymotrypsin with p-nitrophenyl acetate: demonstration of an initial burst
Use of an alternate, poor substrate to change the rate-determining step
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Scheme 2.3
Typical enzyme reaction in which the first step is fast
E•S'
E + P2slow
initial burst
fast
+ P1
E•SE + S
P1 = O NO2 P2 = CH3COO
For para-nitrophenylacetate
E•P2
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Scheme 2.4
common acyl intermediate
Enzymatic rates - same
Nonenzymatic rates - different
PhCH CH C
O
OX
O
PhCH CH C
O
HOXO
2.2 2.3
+
Evidence for formation of an acyl intermediate
Reaction of -chymotrypsin with aryl cinnamate esters
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14CH3C
O
O NO2
O
14CH3CO
O
O
14CH3C
O
O
2.5
2.4
2.6
O NO2
H2O
Scheme 2.5
To demonstrate covalent intermediate:
pH 5 pH 8
stops here
kinetically competent
Formation of an acyl intermediate in the reaction catalyzed by -chymotrypsin
below pH optimum for
catalysis
pH optimum
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excess substrate
Fraction Number
RadioactivityAbs280
( ) ( )
Figure 2.3
Gel Filtration
(aromatic aminoacids in enzyme)
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Scheme 2.6
reactivated enzyme
To support formation of acetylchymotrypsin
Reactivation of acetylchymotrypsin by hydroxylamine
14CH3CO
2.5
14CH3C
O
2.7
NHOHOH..
HONH2
O
Isolate and characterize
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Rate of base hydrolysis of acetylchymotrypsin denatured by 8 M urea is identical to rate of base hydrolysis in 8 M urea with a model compound, O-acetylserinamide
H3C O
O
NH3+
O
NH2
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Scheme 2.7
affinity labeling agent
O OP
O
F
OO
P OO
O
2.8
2.9
Reaction of -chymotrypsin with an organophosphofluoridate affinity labeling agent
To show involvement of a serine residue at the active site
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Scheme 2.8
Affinity labeling agent
substrate protection
E•S
-S + S
E–IE•IE + I
Kinetics of affinity labeling of enzymes
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• Irreversible inhibitors exhibit time-dependent inhibition
Reaction after E•I complex formation is rate limiting; therefore, time
dependent
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Figure 2.4
Enzyme Inactivation
With [32P] get 1 equiv 32P bound to enzyme;
6 N HCl at 110 °C, 24 h gives [32P]phosphoserinePeptidase hydrolysis gives [32P]peptide containing modified Ser-195.
P
F
OOO
Correlation between loss of enzyme activity and incorporation of radioactivity during enzyme inactivation
loss of enzyme activity and incorporation of radioactivity correspond (1 : 1 inactivator : enzyme)
5000
0
100
0
% Enzyme Activity
Radioactivity(dpm)
Time
50 ( )( )
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substrate inactivator (TPCK)
With [14C]TPCK get 1 equiv. [14C] bound; pepsin hydrolysis gives a [14C] peptide with His-57 modified
CH2 CH
NH
SO2
C
CH3
CH2 CH
NH
SO2
C
CH3
OCH3
O O
CH2Cl
2.11 2.12
Evidence for Histidine Participation
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-chymotrypsin
(side reaction) (S)-N-Ac-L-Ala-L-Phe
(S)-N-Ac-L-Ala-L-Phe
Cl
CH3H
2.13
Mechanism of inactivation of -chymotrypsin by -chloromethyl ketones
OH
CH3
H
Evidence against a single SN2 reaction
Same stereochemistryas 2.13
No hydrolysis product in absence of enzyme(nonenzyme control)
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Scheme 2.10
R
O
CH3R
Cl
O
O
O
R
OSer
SerSer O H
OH
R
O
OSer
Cl
HH
CH3 CH3H
H OH
B:
H
CH3R
O
CH3
OH
H
Ser OH
fast
195195 195
195
inversion
inversion
195
2.14 2.15
2.162.17
B:
Double inversion mechanism for inactivation of serine proteases by -chloromethyl ketones
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Scheme 2.11
inversion of configuration
overall retention of configuration
Three possible mechanisms for inactivation
of -chymotrypsin by -chloromethyl ketones
N
HN
O
ClR
N
N
O
N
HN
O
N
HN
O
ClR
N
HN
Cl
R
O
N
N
R
O
O
O
N
HN
O
ClR
H
CH3
HCH3
HCH3
HCH3
HCH3
H
CH3
N
HN
Cl
R
O
O
HCH3
HH3C
N
HN
OO
H
H3C
R
R
N
HN
OH
O
H
H3C
R
R
EE:
E
E
O—H
E
-E
1)
2)
3) E
O—H
E
EE
2.18
2.19
OH OH
BO
B
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HN H
Cl
CH3
O
OPh
AcNH
CH3
2.20
-Chymotrypsin was inactivated by 2.20, and X-ray crystal structure showed His-
57 alkylated with stereochemistry retained
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acetyl-serine model
General base catalysis by imidazole solvent 2H isotope effect 2-3
C
O
OCH3 CH2 CH C
NH
C
CH3
O
2.21
O
NH2
Evidence for Deacylation Mechanism
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Ph O
OHN N
Ph O
O
NH
N
2.22 2.23
Ser mimic His mimic
kH2O/kD2O = 3
Addition of PhCOO- as a model of Asp-102 increases rate 2500 fold
not active
Model study for deacylation step
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Scheme 2.12
Improved model 1/18 rate of chymotrypsin
general base catalysis
Ph O
ON N H
O
OHO H
Ph O
OHN N
O
OHOH Ph
OH
O
HN N
O
OH
O
2.242.25
Chemical model for the deacylation step in -chymotrypsin
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Table 2.1. Rate of Deacylation of Model Compounds Compared to Cinnamoyl-a-chymotrypsin
Compound Relative rate ( krel)
Ph O
O
chymotrypsin 1.0
2.22 2.6 x 10 -7
2.22plus benzoate ion
6.6 x 10 -4
2.24 5.6 x 10 -2
Ph O
OHN N
2.22
Ph O
ON N H
O
OHO H
2.24
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Scheme 2.14
Aspartate Protease
Note: General acid-base catalysis, not covalent catalysis
Proposed mechanism for HIV-1 protease
NH
HO
N
OC
H
HO
O
Asp25
H
OH
O
Asp25'
O
NH
HO
N
O
H
H
O
O
Asp25
H
OH
O
Asp25'
O
NH
HO
N
O
H
H
O
O
Asp25
H
O
HO
Asp25'
O
N
HO
N
O
H
O
O
Asp25
H
O
O
Asp25'
O
H
H
NH
HO
N
O
H
O
O
Asp25
OH
O
Asp25'
O
H
H
O
R'
C
O
R'C
O
R'
C
O
R'C
O
R'
- -
+
-
-
δ
δ -δ
-δ..
+
--
..RR R
RR
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Affinity labeling agent for CPA
labels Glu-270
CH2 CH COOH
NMe
CO
CH2Br
2.30
Carboxypeptidases (an exopeptidase)
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Scheme 2.15
Zn++ is a cofactor
C NH
CHCOO
O
R
R
HOH
Glu270 COO-
Tyr248OH
Zn++
R O
O
Zn++
Tyr248-O
Glu270 COO
NH2 CH COO-
RH
HArg145+
General base catalytic mechanism for carboxypeptidase A
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Scheme 2.16Not detected or trapped
C NH
CHCOO
O
R
R
C O-Glu270
O
Tyr248OH
Zn++ O
CR
O
COGlu270
Zn++
O
CR
Zn++
NH2 CH COO-
R
Glu270 COO-
O-
Arg145+
H2O
Nucleophilic mechanism for carboxypeptidase A
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Principle of Microscopic Reversibility
For any reversible reaction, the mechanism inthe reverse direction must be identical to thatin the forward reaction (only reversed)
This can be a valuable approach to study enzyme mechanisms.
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Scheme 2.17
R C
O
18O-
Glu CO
O-
R C
O
NH CHCO2-
R'
H2N CH
- H218O
CO2-
R'
Reverse of the general base mechanismReverse of general base catalytic reaction of carboxypeptidase A in the presence of H2
18O
Requires amino acid to release H2
18O
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Scheme 2.18
Reverse of the nucleophilic mechanism
R C
O
18O-
Glu C
O
O-
R C
O
O C
O
GluR C
O
NH CHCO2-
R'H2N CH
CO2-
R'
- H218O
Reverse of nucleophilic catalytic reaction of carboxypeptidase A in the presence of H2
18O
Does not require amino acid to release H2
18O
Found amino acid is required for H218O release
(general base mechanism)
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Scheme 2.19
From Crystal Structure of Ketone
Alternative mechanism for carboxypeptidase A on the basis of the X-ray structure with a ketone bound
270Glu O
O
H
O
Zn++
R
CHCOO-
:NH
O
R'
H3N127Arg
H
270Glu O
O
H
H
R
CHCOO-
:NH
C O-
R'H3N127Arg
O
Zn++
270Glu O
OR
CHCOO-
NH3+
O CO
R'Zn+++
+
tetrahedral intermediate
Functions of Zn++ Cofactor• Coordinate to H2O to make it more nucleophilic• Coordinate to carbonyl to make it more electrophilic
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Scheme 2.20
R OR'
O
O H :B
H B
R
O
O HBR
O
O BHH OHB
OH :B
R'OH
RCO2H
H2O
Typical esterase mechanism
Covalent catalytic mechanism
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OCH3
OHB
Me3NCH2CH2—O O
CH3
OB
H
H
Me3NCH2CH2—OH
B:
ester site
+-+
"anionic site"
Me3NCH2CH2OH + CH3COOH+
- +:B
H2O
Scheme 2.21
no anioncluster of aromatic residues instead(cation- complex)
Catalytic triad has a Glu instead of an Asp
Mechanism for acetylcholinesterase
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Favored enantiomer substrate for lipases
Medium Large
H
2.31
R O
O
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Scheme 2.22
O
O H
(1R,2S,5R)-menthyl pentanoate
+
O
O H
(1S,2R,5S)-menthyl pentanoate
lipase
HO H
(1R,2S,5R)-menthol
+
O
O H
(1S,2R,5S)-menthyl pentanoate
An example of the enantioselectivity of lipases/esterases
Useful for chiral resolutions of alcohols
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Catalytic Antibodies (abzymes)
• Antibodies are proteins that scavenge macromolecular xenobiotics
• Form very tight complexes with macromolecule, which causes a cascade of events, leading to degradation of macromolecule
• A catalytic antibody is an antibody that catalyzes a chemical reaction
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Construction of Catalytic Antibodies
• A transition state analogue that mimics the transition state of the desired reaction is synthesized--called a hapten
• Hapten is attached to a carrier molecule capable of eliciting an antibody response--called an antigen
• Antigen injected into a mouse or rabbit
• Monoclonal antibodies (ones that bind to one region of the antigen) are isolated for that antigen
• The monoclonals are tested for catalytic activity
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Transition State Analogue Inhibitor
• Inhibitor molecules resembling the transition-state species should bind to enzyme much more tightly than the substrate
• Therefore, a potent enzyme inhibitor would be a stable compound whose structure resembles that of the substrate at a postulated transition state--a transition state inhibitor
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Development of Catalytic Antibodies
Figure 2.5
R OR'
O
OHR
POR'
O
O
Ester hydrolysisintermediate
"Transition state" mimic
R OR'
O
HO
Comparison of an ester hydrolysis tetrahedral intermediate and a
phosphonate “transition state” mimic
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Ph NH
PO
NHNH
OPh
O-
O O Me
O
NHX
O
O
2.32
mimics tetrahedral intermediate in ester hydrolysis
X = OH haptenX = macromolecule antigen (elicits antibody
response)
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R1 = Bn R2 = HR1 = H R2 = Bn
NH2 O
NH
R1 R2
O
O
O
NH
Me
O
NH
NO2
2.33
Two different monoclonal antibodies raised, each catalyzes hydrolysis of different epimer
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Aminations
Table 2.2. Types of Reactions Catalyzed by Glutamine-Dependent Enzymes
1)C OX C NH
2+
"NH3
"+
-
OX
2)
X
NH2
+ "NH3
"
3)C O
-
O
C NH2
O
"NH3
"
ATP
+
4)C
O
C
NH2
"NH3
"
ATP
+
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Scheme 2.23
Glutaminase activity (generation of NH3)
• Free NH3 is toxic to cell - this protects cell from NH3
• NH3 can be substituted for Gln, but Km 102-103 higher
A covalent catalytic mechanism for the “glutaminase” activity of glutamine-dependent enzymes
NH2H3N
-OOCO
X
H B+
NH2
H3N
-OOC O
X
H:B
XH3N
-OOCOH B+
XGlu
Aminated product
+ "NH3"
acceptor
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Scheme 2.24
Evidence for covalent catalysis
X
O O
NHOHXH
NH3+
-OOC-OOC
NH3+
2.352.34
NH2OH
Evidence for -glutamyl enzyme intermediate in glutamine-dependent enzyme
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Figure 2.6
NH3+
OOCCl
O
NH3+
OOCNH2
O
2.36
Gln
Comparison of the structure of the -chloromethyl ketone of asparagine
with the structure of glutamine
irreversible inhibitor
substrate
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modify Cys residue
Blocks enzyme reaction with Gln, but not with NH3; therefore 2 binding sites
2.37
O
CCH2 NH2I
N
O
O
Et
2.38
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-OOCCH
+NH3
O
N N+ _
2.39
-OOCO CH
+NH3
O
N N
_+
2.40
Mechanism-based inactivators of Gln-dependent enzymes
Mechanism-based inactivator• Unreactive compound whose structure resembles the substrate (or product) for an enzyme• Acts like a substrate and is converted into a species that inactivates the enzyme• Cannot escape enzyme until it inactivates it
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Scheme 2.26
partition ratio = 70 (d/c)
When R contains 3H, ratio of 14C/3H remains constant after inactivation
Mechanisms for inactivation of glutamine-dependent enzymes by -diazoketones
R 14CH
O
N N
H B+
R 14CH2
O
N N
X
R 14CH2
O
X
R 14CH2
O
N NX
XR X
O
R14CH2
O
XY
R 14CH2
O
YX
+ _+
ab
a
b
+
Glu or Ser PhCO214Me 14MeOH+
(E I) (E I')
a
2.39/2.40 2.41 2.42
2.432.44 2.45
c
cd
d
d
+ +
2.462.47
c
b
H2O14CH2N2
PhCO2H
-N2
-N2
H2O
Therefore, 2.39 is responsible for inactivation, not diazomethane (would only be 14C labeled)
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Scheme 2.25
partition ratio = k3/k4
Ideally would be 0
k1
k-1
k3
k2 k4
E + I'
E • I' E - I''E • IE + I
Kinetics for mechanism-based inactivation
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Acceptor reactions are mostly ATP-dependent
Scheme 2.27
An example where no ATP is required
5-phosphoribosyl-1-diphosphate amidotransferase
Amination reaction catalyzed by glutamine phosphoribosyldiphosphate amidotransferase
O
HO OHOP2O6
3-
=O3PO O
HO OH
NH2=O3PO
+ P2O74-
2.48-configuration β-configuration
+ ":NH3"
good leaving group
SN2-like reaction
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What happens when NH3 is added to a carboxylic acid?
Scheme 2.28
+ PhCO2 NH4
+PhCO2H NH3
Function of ATP
Reaction of ammonia with benzoic acid
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Scheme 2.29
ATP Chemical Equivalents
R Cl
O
R NH2
O
R NH2
O
O
O O
HO
O
R O
O O
+ +-SO2
+
2.49
+
2.50
SOCl2HCl
RCO2H
RCO2H-HCl
NH3
NH3-CH3COOH
Activation of carboxylic acid with thionyl chloride and acetic anhydride
ATP acts like SOCl2 or Ac2O
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Figure 2.7
Requires Mg2+ for activity (coordinates to phosphate oxyanions)
Electrophilic sites on ATP
O
HO OH
N
O P
O
O
PO
O
O
PO
O
O
O
CH2 N
Nu-
β
-3 kcal/mol-7 kcal/mol
phosphoesterphosphoric acidanhydride
5'
ATP
N
N
NH2
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Nu P
O
O
PO
O
O
O-
Nu P
O-
O
O
Nu P
O
O
PO
O
O
O Ado
Nu P
O
O
O Ado + PPi
or+ Pi
+ ADP
+ AMP
NuH + Pi
−
β−
−
NuH + PPiNuH + ADP
NuH + AMP
H2O
H2O
Figure 2.8
Products of reaction of nucleophiles at the -, β-, and -positions of ATP
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Scheme 2.30
Asp COOH Gln C
O
NH2 Asn C
O
NH2 Glu COOH+
Mg•ATP Mg•AMP + PPi
+
Reaction Catalyzed by Asparagine Synthetase
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Scheme 2.31
Two possible modes of attack to give AMP + PPi
Activation of aspartate by ATP followed by reaction with ammonia generated from glutamine
Asp C O
O C AMP
O
C PPi
O
Asp
Asp
PPi+
+
.
PPi
+
or Asn + AMP +
-attack
β-attack
ATPMg
AMP
NH3
Gln
-Glu
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Scheme 2.32
[18O] AMP
[18O] PPi
*experimental result
Use of 18O-labeled aspartate to differentiate attack at the - or β-positions of ATP
AspC18O
O
AspC 18O
O
-O P O P O P O Ado
O
O-
O
O-
O
O-
-O P O P O P O Ado
O
O-
O
O-
O
O-
AspC 18O
O
P OAdo
O
O-
AspC 18O
O
P O P O-
O
O-
O
O-
C
O
-18O P O P O-
O
O-
O
O-
Mg++
Mg++
-18O P OAdo
O
O-
Asn NH2
C
O
Asn NH2β-attack
-PPi
-AMP
-attack
+
+
NH3
NH3
*
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Scheme 2.33FGAR
Reaction catalyzed by formylglycinamide ribonucleotide (FGAR) aminotransferase
Important enzyme in purine biosynthesis
O
HO OH
NH=O3PO
O
HNOHC
O
HO OH
NH=O3PO
NH
HNOHC
2.52
+ Mg•ADP+ Gln + Mg•ATP
2.51
+ Pi + Glu
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Scheme 2.34
Use of 18O-labeled FGAR to differentiate attack at the - or β-positions of ATP
-O P
O
O-
PO
O
O-
O
O
HO OH
NH=O3PO
NH2
HNOHC
P Ado
Mg++
NH
OHCN
R
18O
H
NH
HN
R
18O
OHC
P
O-
O
O-
18O P
O-
O
O-
NH
: NH2HNOHC
R
18O P
O
O
O
ADP
+
O
O-
Gln
-Glu
:NH3
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Scheme 2.35
Partial exchange reaction - a way to detect intermediates in multi-step reactions
Therefore attack occurs at the -position
Use of AD32P in a partial reaction to test for reversibility of FGAR aminotransferase and test whether ADP or Pi is
released during the reaction (Gln omitted)
-O P
O
O-
PO
O
O-
O P
Mg++
NH
OHCN
R
O
H
NH
HN
R
O
OHC
P
O-
O
O-
O
O
OAdo
32PO
O
O
O P
Mg++
O
O
OAdo
-O P
O
O-
32PO
O
O-
O P
Mg++
O
O
OAdo
NH
OHCN
R
O
H
NH
HN
R
O
OHC
P
O-
O
O-
2.53(ATP)
2.53
+
+
(AD32P)
(AT32P)
+
ADP
Forwardreaction
Reversereaction
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Scheme 2.36
If β-attack had occurred:
partial exchange w/ 32Pi
-O P
O
O-
PO
O
O-
O P
Mg++
NH
OHCN
R
O
H
NH
HN
R
O
OHC
P
O-
O
O
O
O
OAdoP OAdo
O
O-+
(ATP)
Pi
Pi
Pi
Outcome if FGAR aminotransferase proceeded by formation of ADP phosphate ester
No AT32P would have been formed with added AD32P because ADP would not be an intermediate
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If neither experiment leads to incorporation of 32P into the ATP, it does not mean that neither intermediate is formed
• Assumed enzyme followed an ordered mechanism and that the first partial reaction could proceed in the absence of glutamine: Maybe enzyme needs the glutamine to be bound before activation occurs Binding of glutamine may cause a conformational change that sets up binding site for FGAR and ATP
• Another potential problem - ADP generated in the first partial reaction may bind very tightly, so dissociation and exchange with AD32P do not occur
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Aspartate as the NH3 source
Scheme 2.37
-attack
Mechanisms for the reactions of argininosuccinate synthetase, an aspartate-dependent enzyme, and argininosuccinate lyase.
ATP is abbreviated as POPOPOAdo :NH2
C 18O
NH
CH2
CH2
CH-OOC
NH3+
NH3 CH
CH2
COO-
COO- NH2+
NH2
NH
CH2
CH2
CH-OOC
NH3+
COO-
-OOC
NH2
C 18OPOAdo
NH
COO-
NH3+
NH2 C
H
CH2COO-
COO-
:B Enz
NH2+
NH
NH
NH3+-OOC
CH
COO-
CHCOO-
H
(argininosuccinate lyase)
1. argininosuccinate synthetase +
2.55
Mg•AMP + PPi+
++
PPi
POPO-POAdo
Mg•ATP
2.54 2.56
2.572. argininosuccinate lyase
(argininosuccinate synthetase)
AMP(18O)
(18O)
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Figure 2.9
Phosphorylations
R O P
O
O-
O-
X PO32- Y PO3
2-
R O P
O
OR'
O-
H2O ROH + Pi
+ X-
H2O ROPO32- + R'OH
phosphatase
phosphodiesterase
kinase
electrophile nucleophile enzyme family reaction type
+
+
+
products
transfer
hydrolysis
hydrolysis
Y-
Comparison of the reactions of a phosphatase, a phosphodiesterase, and a kinase
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Scheme 2.38
metaphosphate
R O P
O
O-
O-
B+ H
HO H
:B
Enz X EnzX P
O
O-
O-
R O P
O
O-
O-
B+ H
R OPO32-
HO H
:B
R O P
O
O-
O-
B+ H
HO H
:B
P
OO
O-
HO H:B
R O P
O
O-
O-
B+ H
P
OO
O-
EnzX
EnzX P
O
O-
O-
ROH + Pi
+ ROH
Enz-X + Pi
HO H
:B
+ General Acid-Base Catalysis-associative
Covalent Catalysisassociative+
ROH + Pi
R O P
O-
O-O-
PiROH +General Acid-Base Catalysis-dissociative
ROH +
B+ H
Enz-X + Pi
Covalent Catalysisdissociative
O1)H
2)
H
1)
B
2)
‡
SN2
A
B
C
Three general mechanisms for phosphatases
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Phosphatases
How would you test mechanism?• Mechanism C differentiated from mechanisms A and B
by incubation with H218O
• Associative and dissociative mechanisms are differentiated
by secondary kinetic isotope effects:
Substitution of the phosphate oxygen atoms with 18O gives slower reaction in an associative mechanism (lower bond order; 18O-P is stronger than O-P bond; normal secondary isotope effect), but a faster reaction in a dissociative mechanism (18O=P is higher bond order; more stable transition state; lower activation energy; inverse secondary isotope effect)
•Associative mechanism gives inversion of stereochemistry
about the phosphorus atom, but this may or may not occur
with a dissociative mechanism
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Scheme 2.39
H218O adds to P
2.58 + [14C]2.59 [14C]2.58
2.58 + 32Pi No [32P]2.58
[32P]2.58 [32P]peptide
G 6-P’ase
phenol tryptic
quench digestion
KOH[32P]His
G 6-P’ase
G 6-P’ase
digestion
Therefore phosphoenzyme formed reversibly with release of glucose followed by irreversible hydrolysis of phosphoenzyme to Pi
Reaction catalyzed by glucose 6-phosphatase
O OHOH
OH
HO
O P
O
O
O
O OHOH
OH
HO
OH
+ H2O + Pi
2.58 2.59
(excludes SN2)
Reversible reaction
Irreversible Pi formation
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Scheme 2.40
Common Mechanistic Feature (partial reaction) of the Enolase Superfamily
Common active site structural feature to catalyze a variety of different reactions in different enzymes.
R O
O-R' H
B:
R O-
O-R'
1,1-proton transfer (racemization)
β-elimination of OH-
β-elimination of NH3
β-elimination of R"COO-
M2+ M2+
Superfamilies of Enzymes
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Scheme 2.41
Dissociative covalent catalytic mechanism for VH1 dual-specific Tyr phosphatase
(also hydrolyzes phosphoserine and phosphothreonine residues)
pKa 5.6
Expected stereochemistry of phosphate?
Mechanism for the reaction catalyzed by human dual-specific (vaccinia H1-related)
protein tyrosine phosphatase
92Asp
OOH
O P
O
O-
O-124Cys-S
OH
92Asp
OO-
124CysSP
O
O-
O-
H
O
H
92Asp
OOH
124Cys-SP
O
OO-
92Asp
OO-
HPO4-2
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Figure 2.10
Associative mechanism - favored by metal ions
Ser/Thr phosphatase PP1
Metal ions make the H2O more nucleophilic and the phosphate more electrophilic
Stereochemistry?
(a) Molecular model of the active site of protein serine/ threonine phosphatase PP1 with tungstate ion (WO4) bound; (b) Schematic of the catalytic mechanism based on the crystal structure and kinetic studies
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R
O
P O-O
O
CH2
O
OO
C
P O-O
O
CH2
O
OHO
A
P O-O
OR'
B+ H
:B
O
OO
C
PO O-
O
OHO
AHO
P
B:H OH
B+H
H
-O O
OR'
R
O
P O-O
O
CH2
O
OH
C
R
O
P O-O
O
CH2
+
2.62
2-O3PO
Scheme 2.42
Phosphodiesterases
12His
119His
General acid/base-catalyzed reaction for ribonuclease A
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Kinases
• Transfer the -phosphoryl group of nucleoside triphosphates (originally only ATP) to an acceptor
• Now generalized to reactions at the -, β-, or -position of any nucleoside triphosphate
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Kinases
Scheme 2.44 phosphoenolpyruvatePEP
trapped w/Br2
No evidence for a phosphoenzyme intermediate
In the presence of an ATP mimic in 3H2O, 3H is incorporated into pyruvate
H2C
H
C
O
COO- CH2 C
O
COO- CH2
OPO3=
COO-
2.68
+ ADPP-O-P-O-P-O-Ado
2.66 2.67
HB: B:
Mechanism for pyruvate kinase (ATP is abbreviated POPOPOAdo)
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Scheme 2.45
CH3C
O
O CH3C
O
OPOAdo CH3C
O
SCoA P-O-P-O-P-O-Ado
PPi
+ AMP
N
N N
N
O
HO OPO3=
CH2 OP
O
O-
OP
O
O-
OCH2 C
CH3
CH3
C
OH
H
C
O
NH CH2 CH2 C
O
NHCH2CH2SH
NH2
2.69
+ CoASH
CoASH
Mechanism for acetyl-CoA synthetase (ATP is abbreviated POPOPOAdo)