Transcript 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.

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
Reaction catalyzed by peptidases
S2
S1
S1'
S2'
P2
P1
P1'
P2'
O
+
NH3
CH
O
H
N
C
CH
R1
O
C
NH
R2
CH
C
O
NH
R3
CH
C
N
H
R4
scissile bond
H2O
O
+
NH3
CH
C
O
NH
R1
Scheme 2.1
CH COOR2
+
+
NH3
CH
R3
C
O
NH
CH C N
H
R4
Classifications of peptidases
exopeptidase
(aminopeptidase)
exopeptidase
(carboxypeptidase)
endopeptidase
O
O
O
+
NH3
CH
R1
Figure 2.1
C
NH
CH C
R2
NH
CH C
R3
NH
CH COOR4
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
O
+
NH3
CH
R1
C
O
NH
CH C
R2
P1
O
NH
CH C
R3
NH
CH COO
R4
Endopeptidase
+
O
R
Mechanism for
-chymotrypsin
showing
catalytic triad
Ser195
+
C
X
O
acylation
O
R
H
Ser
C
X
O
H
N
N
-O
H
N
Asp102
C
N
H
-O
C
Asp
O
O
His57
His
-XH
+
+
O
O
O
Ser
C
R
O
deacylation
OH
C
R
Ser
OH
H
H
N
N
H
-O
C
Asp
O
N
N
H
-O
C
Asp
O
His
His
acyl intermediate
+
R
Ser195 OH
COOH
N
N
H
-O
C
O
His57
Asp102
Scheme 2.2
Evidence for Acyl Intermediate
Reaction of chymotrypsin with p-nitrophenyl
acetate: demonstration of an initial burst
Use of an alternate, poor substrate to
change the rate-determining step
Figure 2.2
O
CH3C
O
NO2
2.1
A400 nm
(Release of
-O
NO2 )
steady state phase
initial burst phase
corresponds to 1 equiv
per equiv of enzyme
Time
Typical enzyme reaction in which
the first step is fast
E+S
E•S
fast
E•S'
initial burst
+ P1
slow
For para-nitrophenylacetate
P1 =
Scheme 2.3
O
NO2
P2 = CH3COO
E•P2
E + P2
Evidence for formation of an acyl intermediate
Reaction of -chymotrypsin with aryl cinnamate esters
common acyl
intermediate
O
O
X
PhCH
CH C O
PhCH
CH C
O
2.2
X
+
HO
O
2.3
Scheme 2.4
Enzymatic rates - same
Nonenzymatic rates - different
To demonstrate covalent intermediate:
Formation of an acyl intermediate in the
reaction catalyzed by -chymotrypsin
O
O
14CH C
3
O
O
NO2
NO2
pH 5
2.4
14CH C
3
O
2.5
Scheme 2.5
below pH
optimum for
catalysis
stops
here
O
O
H2O
pH 8
O
14CH C
3
pH optimum
O
2.6
kinetically
competent
Gel Filtration
Abs280
(
Radioactivity
)
(
(aromatic amino
acids in enzyme)
excess
substrate
Fraction Number
Figure 2.3
)
Reactivation of acetylchymotrypsin
by hydroxylamine
To support formation of acetylchymotrypsin
..
HONH2
14CH C
3
O
2.5
O
14CH C
3
NHOH
O
2.7
Isolate and characterize
Scheme 2.6
OH
reactivated
enzyme
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
O
H3C
O
O
NH2
NH3+
Reaction of -chymotrypsin with an
organophosphofluoridate affinity labeling agent
To show involvement of a serine residue at
the active site
affinity labeling agent
O
O
P
O
O
F
O
O
P
O
2.8
2.9
Scheme 2.7
O
Affinity labeling agent
Kinetics of affinity labeling of enzymes
E+I
substrate
protection
-S
+S
E•S
Scheme 2.8
E•I
E–I
• Irreversible inhibitors exhibit time-dependent
inhibition
Reaction after E•I complex formation is
rate limiting; therefore, time dependent
Enzyme Inactivation
Correlation between loss of enzyme activity and
incorporation of radioactivity during enzyme inactivation
100
5000
loss of enzyme activity and
incorporation of radioactivity
correspond
(1 : 1 inactivator : enzyme)
% Enzyme
Activity
(
)
50
0
(
)
Figure 2.4
0
Time
O
With [32P]
Radioactivity
(dpm)
O
O
P
get 1 equiv 32P bound to enzyme;
F
6 N HCl at 110 °C, 24 h gives [32P]phosphoserine
Peptidase hydrolysis gives [32P]peptide containing modified Ser-195.
Evidence for Histidine Participation
O
CH2 CH
NH
C OCH3
O
CH2 CH
NH
SO2
SO2
CH3
CH3
C CH2Cl
2.11
2.12
substrate
inactivator
(TPCK)
With [14C]TPCK get 1 equiv. [14C] bound; pepsin hydrolysis
gives a [14C] peptide with His-57 modified
Mechanism of inactivation of chymotrypsin by -chloromethyl ketones
CH3
H
(S)-N-Ac-L-Ala-L-Phe
2.13
Cl
-chymotrypsin
(side reaction)
(S)-N-Ac-L-Ala-L-Phe
No hydrolysis product in absence of enzyme
(nonenzyme control)
Evidence against a single SN2 reaction
CH3
H
OH
Same stereochemistry
as 2.13
Double inversion mechanism for inactivation of
serine proteases by -chloromethyl ketones
O
Ser
O
O
CH3
R
195
H
H
CH3
fast
R
Cl
O H
195
O
R
195
Cl
Ser
inversion
CH3
H
OH
O
Ser
H
2.14
B:
2.15
B:
inversion
O
R
195
Ser
Scheme 2.10
OH
O
H
H
CH3
OH
2.17
R
195 Ser
CH3
O
OH
2.16
H
N
Three possible
mechanisms for inactivation
of -chymotrypsin by
-chloromethyl ketones
Scheme 2.11
N
N:
1) E
O
R
inversion of
configuration
O
Cl
R
OH
H
N
H
N
2.18
N
N
N
H
R
-O
Cl
O—H
CH3
E
H
CH3
O
B
O
N
N
E
H
O—H
H
N
H3C
CH3
O
CH3
Cl
H
N
E
Cl
R
CH3
H
R
O
H
N
R
O
R
H
N
O
N
E
Cl
H
3) E
H
CH3
OH
2) E
N
E
H
CH3
R
O
O
O
B
H
N
H
N
N
H3 C
overall retention
of configuration
E
H3C
H
E
N
H
R
O
OH
2.19
R
O
O
-Chymotrypsin was inactivated by 2.20,
and X-ray crystal structure showed His57 alkylated with stereochemistry retained
CH3
H
N
AcNH
O
O
CH3
H
Cl
Ph
2.20
Evidence for Deacylation Mechanism
O
O
CH3 C
O
CH2
CH
C
NH2
NH
C
2.21
O
CH3
General base catalysis by imidazole
solvent 2H isotope effect 2-3
acetylserine
model
Model study for deacylation step
Ser mimic
N
His mimic
Ph
O
O
Ph
HN
N
H
O
N
O
2.22
kH2O/kD2O = 3
2.23
not active
Addition of PhCOO- as a model of Asp-102 increases
rate 2500 fold
Chemical model for the deacylation step
in -chymotrypsin
1/18 rate of chymotrypsin
Improved model
Ph
O
Ph
O
H
HO
N
N H
O
O
HN
O OH
O
OH
N
Ph
OH
O
HN
N
OH
O
O
O
2.24
2.25
general base catalysis
Scheme 2.12
Table 2.1. Rate ofDeacylati on of Model Compounds Compared
Cinnamoyl
to
-a-chymotrypsi n
Compound
Ph
Relative r ate (k r el)
O chymot ryps in
1.0
O
2.22
2.6 x 10-7
2.22
plus benzoate ion
6.6 x 10-4
2.24
5.6 x 10-2
Ph
Ph
O
O
O
O
HO H
HN
2.22
N
N
N H
O
O
2.24
Aspartate Protease
Proposed mechanism for HIV-1 protease
HO
HO
HO
H
..
N
R N
H
H
R'
O
H
O
O
O
Asp25
H
C
O
R N
H
 -O
O
H
 -O
H
O
O
Asp25'
H
+
N
O
C
H O
R N
H
R'
O
-
H
H
C
O
R'
O
H
O
O
O
Asp25
Asp25
Asp25'
Asp25'
HO
H
R N
H
H
N
C
O
OH
O
R'
H
O
H
O
Asp25
+
N
R N
O
O
Scheme 2.14
H
-O
HO
Note: General acidbase catalysis, not
covalent catalysis
 -O
N
..
O
O H
H
H
O
O
Asp25'
O
Asp25
C
O
O
R'
-O
Asp25'
Carboxypeptidases (an exopeptidase)
Affinity labeling agent for CPA
CH2
CH COOH
NMe
O
C
CH2Br
2.30
labels Glu-270
General base catalytic mechanism for
carboxypeptidase A
Zn++
O
Zn++
Tyr248
H O
-O
O
CHCOO
R
+
Arg145
R
O H
:
R C N
H
NH2 CH COO-
OH
H
Glu270 COO
Glu270 COO-
Scheme 2.15
Zn++ is a cofactor
Tyr248
H
R
Nucleophilic mechanism for
carboxypeptidase A
Zn++
Zn++
R C N
H
Glu270
O
C
O
Scheme 2.16
R
H O
C
Tyr248
O
:
O
Zn++
O
CHCOO
R
O
+
Arg145
C
Glu270
NH2 CH
H2O
O
COO-
R
Not detected
or trapped
R C OGlu270 COO-
Principle of Microscopic Reversibility
For any reversible reaction, the mechanism in
the reverse direction must be identical to that
in the forward reaction (only reversed)
This can be a valuable approach to study
enzyme mechanisms.
Reverse of the general base mechanism
Reverse of general base catalytic reaction of
carboxypeptidase A in the presence of H218O
O
R
C
18O-
OGlu
C
- H218O
R
O
R'
C
NH CHCO2-
O
R'
H2N C CO2H
Requires amino acid
to release H218O
Scheme 2.17
Reverse of the nucleophilic mechanism
Reverse of nucleophilic catalytic reaction of
carboxypeptidase A in the presence of H218O
O
R
C
18O-
OGlu
C
O
-
H218O
R
C
R'
H2N C CO2H
O
O
O
C
O
R
C
R'
NH CHCO2-
Glu
Does not
require amino
acid to release
H218O
Scheme 2.18
Found amino acid is required for H218O release
(general base mechanism)
From Crystal Structure of Ketone
Alternative mechanism for carboxypeptidase A on the
basis of the X-ray structure with a ketone bound
270
O
Glu
O
R
Glu
270
O
CHCOOH
:NH
Scheme 2.19
:NH
O
+
Glu
270
H3N Arg
CHCOONH3+
O
C O-
Zn++
127
R
O
O
O
R'
Zn++
O
CHCOO-
H
O
H O
R
H
+
H3N127Arg
R'
Zn++
tetrahedral intermediate
Functions of Zn++ Cofactor
• Coordinate to H2O to make it more nucleophilic
• Coordinate to carbonyl to make it more electrophilic
C
R'
Typical esterase mechanism
O
R
OR'
O H
O
O
R'OH
H B
R
H2O
O
HB
B
R
O
H OH
H B
:B
RCO2H
OH
Scheme 2.20
Covalent catalytic mechanism
:B
Mechanism for acetylcholinesterase
Me3NCH2CH2—O O
+
H
CH3
B+
:B
H
O
"anionic
site"
ester
site
Me3NCH2CH2—OH
B:
O
O
+
HB
CH3
H2O
+
no anion
Me3NCH2CH2OH + CH3COOH
cluster of aromatic
Scheme 2.21
residues instead
(cation- complex)
Catalytic triad has a Glu instead of an Asp
Favored enantiomer substrate for lipases
O
R
O
H
Large
Medium
2.31
An example of the enantioselectivity of
lipases/esterases
O
O
O H
O
lipase
+
(1R,2S,5R)-menthyl pentanoate
HO H
O H
(1S,2R,5S)-menthyl pentanoate
O H
+
(1R,2S,5R)-menthol
(1S,2R,5S)-menthyl pentanoate
Scheme 2.22
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 transitionstate 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
Comparison of an ester hydrolysis
tetrahedral intermediate and a
phosphonate “transition state” mimic
HO
OH
OR'
R
O
OR'
R
OR'
R
P
O
O
Ester hydrolysis
intermediate
Figure 2.5
O
"Transition state"
mimic
mimics tetrahedral intermediate
in ester hydrolysis
O
Ph
O
Ph
O
O
N
H
P
O
O-
NH
Me
NH
NH
X
O
O
2.32
X = OH
hapten
X = macromolecule
antigen (elicits antibody
response)
Two different monoclonal antibodies raised,
each catalyzes hydrolysis of different epimer
NH2
O
O
R1 R2
O
NH
R2 = H
R2 = Bn
Me
NH
NH
O
R1 = Bn
R1 = H
NO2
O
2.33
Aminations
Table 2.2.
1)
Types of Reactions Catalyzed by Glutamine-Dependent Enzymes
C
OX
+
C
"NH3"
NH2
NH2
2)
+
"NH3"
X
3)
C
O-
+
"NH3"
ATP
O
4)
C
O
C
O
+
ATP
"NH3"
C
NH2
NH2
+
- OX
Glutaminase activity (generation of NH3)
A covalent catalytic mechanism for the “glutaminase”
activity of glutamine-dependent enzymes
O
H
H
B+
-OOC
-OOC
H B+
O
NH2
NH2
H3N
:B
H3N
-OOC
O
X
X
H3N
:
X
H 2O
:
+ "NH3"
X
Glu
acceptor
Scheme 2.23
• Free NH3 is toxic to cell - this
protects cell from NH3
• NH3 can be substituted for Gln,
but Km 102-103  higher
Aminated
product
Evidence for covalent catalysis
Evidence for -glutamyl enzyme intermediate in
glutamine-dependent enzyme
NH3+
NH3+
X
-OOC
2.34
O
Scheme 2.24
NH2OH
NHOH
-OOC
O
2.35
XH
Comparison of the structure of the
-chloromethyl ketone of asparagine
with the structure of glutamine
NH3+ O
OOC
Cl
irreversible inhibitor
NH2
substrate
2.36
NH3+
OOC
Gln
Figure 2.6
O
O
O
I CH2
C
N Et
NH2
2.37
O
2.38
modify Cys residue
Blocks enzyme reaction with Gln, but not
with NH3; therefore 2 binding sites
Mechanism-based inactivators of
Gln-dependent enzymes
O
O
-OOC
CH
+NH
3
+
N
_
N
-OOC
O
CH
+
N
+NH
3
2.39
2.40
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
_
N
Mechanisms for inactivation of glutaminedependent enzymes by -diazoketones
H B+
b
O
R
CH
14
(E
+
N
O
14
R
b
_
N
I)
a
CH2
O
+
N N
R
a
a
(E
I')
2.41
b
2.42
d
R
14
X
+
N N
CH2
c
d
c
R
+
X
14
CH2
-N2
X
CH2
Y
X
Y
2.45
2.44
partition ratio = 70 (d/c)
O
Glu or Ser
R
14
2.43
d
O
O
c
O
CH2
X
-N2
X
2.39/2.40
Scheme 2.26
14
H2 O
X
R
2.46
+
PhCO2H
14
CH2N2
H2 O
PhCO214Me
+
14
MeOH
2.47
When R contains 3H, ratio of 14C/3H remains constant after inactivation
Therefore, 2.39 is responsible for inactivation, not diazomethane (would only be
14C
labeled)
Kinetics for mechanism-based inactivation
E+I
k1
E•I
k2
E • I'
k-1
k3
Scheme 2.25
E + I'
partition ratio = k3/k4
Ideally would be 0
k4
E - I''
Acceptor reactions are mostly ATP-dependent
An example where no ATP is required
Amination reaction catalyzed by glutamine
phosphoribosyldiphosphate amidotransferase
=O PO
3
=O PO
3
O
OP2O63HO
Scheme 2.27
NH2
+ P2O74-
+ ":NH3"
HO
OH
-configuration
2.48
O
good leaving
group
OH
-configuration
SN2-like reaction
5-phosphoribosyl-1-diphosphate amidotransferase
Function of ATP
What happens when NH3 is added to a
carboxylic acid?
Reaction of ammonia with benzoic acid
PhCO2H
+
NH3
Scheme 2.28
PhCO2 NH4+
ATP Chemical Equivalents
Activation of carboxylic acid with thionyl chloride
and acetic anhydride
O
-SO2
RCO2H + SOCl2
-HCl
R
O
NH3
R
Cl
NH2
+
HCl
2.49
O
RCO2H +
O
O
O
-CH3COOH
R
O
O
O
O
NH3
+
R
2.50
Scheme 2.29
ATP acts like SOCl2 or Ac2O
NH2
HO
Electrophilic sites on ATP
Figure 2.7
O



O
O
O
P
O
O
P
O
O
P
5'
N
O CH2
phosphoric acid
anhydride
N
O
N
N
O
Nu-7 kcal/mol
NH2
HO
OH
-3 kcal/mol
phosphoester
ATP
Requires Mg2+ for activity (coordinates
to phosphate oxyanions)
Products of reaction of nucleophiles at the
-, -, and -positions of ATP
H2O
NuH + Pi
O
Nu
P
O
+ ADP
O
H2O
H2O
O

Nu
P
O
O
O
P
NuH + PPi
NuH + ADP
O
O
O
Ado
+ Pi
O
or
Nu
P
O
O
P
O-
+
AMP
O
O

Nu
P
O
Ado
+
PPi
O
H2O
NuH + AMP
Figure 2.8
Reaction Catalyzed by Asparagine Synthetase
Asp COOH
+
O
O
Gln C NH2
Asn C NH2
Mg•ATP
Scheme 2.30
Mg•AMP + PPi
+
Glu COOH
Two possible modes of attack
to give AMP + PPi
Activation of aspartate by ATP followed by reaction
with ammonia generated from glutamine
Gln
O
-attack
O
Asp C O
+ Mg . ATP
Asp C AMP +
PPi
NH3
or
O
-attack
Scheme 2.31
-Glu
Asp C
PPi
+ AMP
Asn + AMP + PPi
Use of 18O-labeled aspartate to differentiate
attack at the - or -positions of ATP
O
AspC18O
O
-O
O
O
P O P O P O Ado
O-
O-
O-
-attack
-PPi
Mg++
O
O
AspC 18O
O
P OAdo
Asn C NH2
O-
+
-18O
NH3
O
P OAdo
O-
[18O] AMP*
O
O
AspC 18O
-O
O
O
P O P O P O Ado
O-
O-
OMg++
Scheme 2.32
-attack
-AMP
O
AspC 18O
O
P O P OO-
NH3
O
O
Asn C NH2
O-
O
+
-18O
O
P O P OOO[18O] PPi
*experimental result
Reaction catalyzed by formylglycinamide
ribonucleotide (FGAR) aminotransferase
H
OHC N
H
OHC N
O
=O PO
3
O
NH
NH
=O PO
3
NH
O
+ Mg•ADP
+ Gln + Mg•ATP
HO
OH
2.51
FGAR
HO
OH
+
Pi + Glu
2.52
Scheme 2.33
Important enzyme in purine biosynthesis
Use of 18O-labeled FGAR to differentiate
attack at the - or -positions of ATP
Gln
H
O
OHCN
-O
18
O
R
-Glu
ADP
P O P
O-
N
H
O
O
O P Ado
O-
O-
O
H
N
H
OHC N
NH2
O
NH
O
Scheme 2.34
OH
: NH2
O
O
18
18
O
P O
O
R N
H
P OO-
+N
R H
OHC
HO
O
18
Mg++
=O PO
3
:NH3
H
OHC N
P OO-
Partial exchange reaction - a way to detect
intermediates in multi-step reactions
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)
H
ADP
O
OHCN
-O
O
R
P O P
O-
N
H
H
OHC N
O
O
O
O P OAdo
O-
P O-
O
O
+
R
Mg++
O-
N
H
Forward
reaction
2.53
(ATP)
O
-O
OHCN
P O-
O
+N
R H
2.53
O
H
H
OHC N
O +
OO
O
32P
O
O
32P
O P OAdo
OMg++
O
Reverse
reaction
(AT32P)
O P OAdo
O
Mg++
Scheme 2.35
R
N
H
P O
O-
O
O
(AD32P)
Therefore attack occurs
at the -position
If -attack had occurred:
Outcome if FGAR aminotransferase proceeded
by formation of ADP phosphate ester
H
OHCN
O
O
-O
:
R
N
H
P
O-
O
O
P
O-
Mg++
O
O
Pi
OHC
H
N
P OAdo
O
O
O
Pi
+N
R H
P
O-
O
O P OAdo
O-
Pi
(ATP)
Scheme 2.36
partial exchange w/ 32Pi
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
Mechanisms for the reactions of argininosuccinate synthetase,
an aspartate-dependent enzyme, and argininosuccinate lyase.
ATP is abbreviated as POPOPOAdo
:NH2
C
NH2
CH2
18O
NH
NH2+
COO+ NH3
CH
+ Mg•ATP
COO-
CH2
1. argininosuccinate synthetase
NH
+
2. argininosuccinate lyase
CH2
-OOC
CH2
CH2
-OOC
-OOC
CH
NH3+
2.54
COO-
CH
NH3+
-attack
2.57
+ Mg•AMP + PPi
(18O)
2.56
POPO-POAdo
(argininosuccinate synthetase)
:B
PPi
Enz
H
H
:
NH2
C 18OPOAdo
NH
NH2
C
CH2COO-
COO-
CHCOO-
NH2+
NH
NH
CH
COO-
(argininosuccinate lyase)
AMP(18O)
NH3+
COO-
-OOC
NH3+
2.55
Scheme 2.37
Phosphorylations
Comparison of the reactions of a phosphatase,
a phosphodiesterase, and a kinase
electrophile
nucleophile
products
enzyme family
reaction type
ROH + Pi
phosphatase
hydrolysis
O
R
O
P
O-
+
H2O
OR'
+
H2O
+
Y-
OO
R
O
P
ROPO32- + R'OH
phosphodiesterase
hydrolysis
O-
X
PO32-
Figure 2.9
Y
PO32-
+ X-
kinase
transfer
Three general mechanisms for phosphatases
‡
B+
1)
B+
O
H
R
O
P
O-
+
HO
H
B+
H
R
2)
O
O
P
O
P
ROH +
O- HO
O
O
P
H
O H
ROH + Pi
General Acid-Base Catalysisassociative
B
Pi
General Acid-Base Catalysisdissociative
+ ROH
Covalent Catalysis
associative
H
:B
O
O-
:
R
1)
P
O
O-
metaphosphate
H
O
O- O-
O-
B+
O-
:B
O-
A
R
H
+
Enz
X
EnzX
O-
P
O-
OHO
H
:B
B
Enz-X + Pi
B+
2)
H
R
O
O
P
O
O
O-
ROH
O-
+
EnzX
P
O-
O
EnzX
P
Covalent Catalysis
dissociative
O-
O- HO H
:B
Enz-X + Pi
:B
C
R
OPO32-
HO
H
ROH + Pi
SN2
Scheme 2.38
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
Reaction catalyzed by
glucose 6-phosphatase
O
O P O
O
O
HO
OH
OH + H2O
OH
HO
OH
OH
H2
2.58
18O
OH + Pi
OH
O
2.59
adds to P
2.58 + [14C]2.59
2.58 + 32Pi
[32P]2.58
Scheme 2.39
(excludes SN2)
G 6-P’ase
G 6-P’ase
G 6-P’ase
[14C]2.58
No [32P]2.58
phenol
tryptic
quench
digestion
[32P]His
Reversible reaction
Irreversible Pi formation
[32P]peptide
KOH
Therefore phosphoenzyme formed reversibly with release of glucose
followed by irreversible hydrolysis of phosphoenzyme to Pi
Superfamilies of Enzymes
Common Mechanistic Feature (partial reaction)
of the Enolase Superfamily
1,1-proton transfer (racemization)
R
R
O
O-
M2+
M2+
R'
OH
R'
O-
-elimination of OH-elimination of NH3
-elimination of R"COO-
B:
Scheme 2.40
Common active site structural feature to catalyze a
variety of different reactions in different enzymes.
Dissociative covalent catalytic mechanism for VH1
dual-specific Tyr phosphatase
(also hydrolyzes phosphoserine and
phosphothreonine residues)
Mechanism for the reaction catalyzed by
human dual-specific (vaccinia H1-related)
protein tyrosine phosphatase
92Asp
92Asp
92Asp
O
O
H
OH
O-
O-
Scheme 2.41
O-
H
O O-
O
H
-S
HPO4-2
O
P
P
O-
O
O
O
O
O
O-
92Asp
P
S
124Cys
O
OH
O-
124Cys
pKa 5.6
Expected stereochemistry of phosphate?
-S
124Cys
Associative mechanism - favored by metal ions
Ser/Thr phosphatase PP1
Metal ions make the H2O more nucleophilic and the phosphate more
electrophilic
(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
Stereochemistry?
Figure 2.10
Phosphodiesterases
General acid/base-catalyzed reaction for
ribonuclease A
-O
R
R
O
O
-O
P O
R
O
P O
O
-O
O
CH2
C
O
O
CH2
12His
P O
C
O
CH2
O
H B+
O
-O
O
O H
H OH
CH2
O
A
2.62
B:
+
HO
119His
O
-O
O
A
OH
P O
O
R'
2-O PO
3
O-
O
O
B+ H
O
P
:B
P O
O
-O
P O
O
R'
C
OH
Scheme 2.42
OH
Kinases
• Transfer the -phosphoryl group of nucleoside
triphosphates (originally only ATP) to an
acceptor
• Now generalized to reactions at the -, -, or
-position of any nucleoside triphosphate
Kinases
Mechanism for pyruvate kinase
(ATP is abbreviated POPOPOAdo)
B:
B:
H
H
O
H2C
C
OPO3=
O
COO-
2.66
CH2
C
+ ADP
P-O-P-O-P-O-Ado
COO-
2.67
COO-
CH2
2.68
phosphoenolpyruvate
PEP
Scheme 2.44
trapped w/Br2
No evidence for a phosphoenzyme intermediate
In the presence of an ATP mimic in 3H2O, 3H is
incorporated into pyruvate
Mechanism for acetyl-CoA synthetase
(ATP is abbreviated POPOPOAdo)
PPi
O
CH3C O
+ P-O-P-O-P-O-Ado
NH2
N
CH3C OPOAdo
CoASH
O
CH3C SCoA +
CoASH
N
N
O
N
O O
O
CH2 OP OPOCH2
O- O-
HO
OPO3=
Scheme 2.45
CH3 OH
O
C
C NH CH2 CH2 C NHCH2CH2SH
C
CH3 H
2.69
O
AMP