Post on 21-Dec-2015
The Organic Chemistry of Enzyme-Catalyzed Reactions
Chapter 2
Group Transfer Reactions: Hydrolysis, Amination,
Phosphorylation
Hydrolysis Reactions
Amide Hydrolysis
Peptidases (proteases if protein hydrolysis involved) catalyze the hydrolysis of peptide bonds
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
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
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
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
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
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
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
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
excess substrate
Fraction Number
RadioactivityAbs280
( ) ( )
Figure 2.3
Gel Filtration
(aromatic aminoacids in enzyme)
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
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
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
Scheme 2.8
Affinity labeling agent
substrate protection
E•S
-S + S
E–IE•IE + I
Kinetics of affinity labeling of enzymes
• Irreversible inhibitors exhibit time-dependent inhibition
Reaction after E•I complex formation is rate limiting; therefore, time
dependent
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 ( )( )
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
-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)
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
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
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
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
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
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
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
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
Affinity labeling agent for CPA
labels Glu-270
CH2 CH COOH
NMe
CO
CH2Br
2.30
Carboxypeptidases (an exopeptidase)
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
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
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.
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
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)
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
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
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
Favored enantiomer substrate for lipases
Medium Large
H
2.31
R O
O
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
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
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
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
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
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)
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
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
+
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
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
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
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
-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
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)
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
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
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
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
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
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
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
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
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
*
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
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
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
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
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
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)
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
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
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
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
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
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
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
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
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
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)
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)