Alkaline phosphatase mono- and diesterase reactions: Comparative transition state analysis

Article abstract of DOI:10.1021/ja056528r

Enzyme-catalyzed phosphoryl transfer reactions have frequently been suggested to proceed through transition states that are altered from their solution counterparts. Previous work with Escherichia coli alkaline phosphatase (AP), however, suggests that thi

Full text of DOI:10.1021/ja056528r

Published on Web 01/06/2006
Alkaline Phosphatase Mono- and Diesterase Reactions:
Comparative Transition State Analysis
Jesse G. Zalatan† and Daniel Herschlag*,†,‡
Contribution from the Department of Chemistry, Stanford UniVersity,
Stanford, California 94305, and Department of Biochemistry, Beckman Center B400,
Stanford UniVersity, Stanford California 94305
Received September 22, 2005; E-mail: herschla@cmgm.stanford.edu
Abstract: Enzyme-catalyzed phosphoryl transfer reactions have frequently been suggested to proceed
through transition states that are altered from their solution counterparts. Previous work with Escherichia
coli alkaline phosphatase (AP), however, suggests that this enzyme catalyzes the hydrolysis of phosphate
monoesters through a loose, dissociative transition state, similar to that in solution. AP also exhibits catalytic
promiscuity, with a low level of phosphodiesterase activity, despite the tighter, more associative transition
state for phosphate diester hydrolysis in solution. Because AP is evolutionarily optimized for phosphate
monoester hydrolysis, it is possible that the active site environment alters the transition state for diester
hydrolysis to be looser in its bonding to the incoming and outgoing groups. To test this possibility, we have
measured the nonenzymatic and AP-catalyzed rate of reaction for a series of substituted methyl phenyl
phosphate diesters. The values of âlg and additional data suggest that the transition state for AP-catalyzed
phosphate diester hydrolysis is indistinguishable from that in solution. Instead of altering transition state
structure, AP catalyzes phosphoryl transfer reactions by recognizing and stabilizing transition states similar
to those in aqueous solution. The AP active site therefore has the ability to recognize different transition
states, a property that could assist in the evolutionary optimization of promiscuous activities.
The AP active site contains two Zn2+ ions and an arginine
residue that are positioned to interact with the substrate, and a
Introduction
Escherichia coli alkaline phosphatase (AP) catalyzes the
hydrolysis of a broad range of phosphate monoester substrates,
presumably to harvest phosphate for nucleic acids and various
metabolites.1 AP also catalyzes phosphate diester hydrolysis.2
The 1011-fold overall rate enhancement for enzymatic diester
hydrolysis relative to the solution reaction is substantial, but
small in comparison with the >1017-fold rate enhancement for
monoester hydrolysis. The catalytic promiscuity of AP and other
enzymes supports a model for enzyme evolution in which
enzymes that can catalyze alternative reactions with a low level
of activity will have a selective advantage for evolutionary
optimization of that activity, after a gene duplication event.3-8
Furthermore, the existence of evolutionarily related homologues
of AP that preferentially catalyze phosphate diester hydrolysis
implies that the phosphodiesterase activity of AP can be
optimized to levels that are reasonable for biological function.9-11
serine alkoxide that displaces the leaving group in the first step
of the reaction to produce a covalent enzyme-phosphate
intermediate (Scheme 1A).12,13 This intermediate is subsequently
hydrolyzed by water in the second step of the reaction.1 It has
been proposed that active sites containing two metal ions are
well-suited for catalyzing phosphoryl transfer reactions of both
monoesters and diesters.14,15 In the simplest case, one metal ion
activates the incoming nucleophile, and another metal ion
stabilizes charge buildup on the leaving group oxygen, as
proposed for AP-catalyzed phosphate monoester hydrolysis
(Scheme 1A). This mechanism is very similar to that proposed
for phosphate diester hydrolysis catalyzed by Klenow exo-
nuclease and other phosphodiesterases,14,16,17 raising an impor-
tant question: how do these active sites distinguish between
different types of phosphate ester substrates? These distinctions
are especially important for understanding how a two-metal ion
phosphate monoesterase could evolve into a diesterase or vice
versa.
† Department of Chemistry.
‡ Department of Biochemistry.
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A R T I C L E S
Zalatan and Herschlag
Scheme 1. Proposed AP Active Site Interactions of wt AP with a Phosphate Monoester Substrate (A) and of R166S AP with a Phosphate
Diester Substrate (B)a 12,13
a
Reactions are depicted in terms of a transition state representation with partial bond formation and bond cleavage. No information about bond orders
and bond lengths is conveyed in this schematic.
Scheme 2. Nonenzymatic Transition States for Reactions of Phosphate Monoesters and Diesters
According to transition state theory, enzymatic rate enhance-
ments arise from preferential stabilization of the transition state
relative to the ground state.18-22 Consequently, the specificity
of an enzyme for a particular reaction depends on its ability to
recognize and stabilize the transition state for that reaction, and
characterization of the structure of the transition state is a critical
step toward understanding both how enzymes catalyze reactions
in general and how they distinguish between different reactions.
We sought to characterize the transition state for AP-catalyzed
phosphate diester hydrolysis for comparison with previous
characterizations of the transition state for AP-catalyzed phos-
phate monoester hydrolysis.23-28 Phosphate monoesters and
diesters proceed through different transition states in solution,
raising the possibility that AP alters the transition state for
phosphate diester hydrolysis to resemble that for phosphate
monoesters.
the leaving group and little bond formation to the nucleophile
(Scheme 2A).29-33 In contrast, phosphate diester hydrolysis in
solution proceeds through a tighter transition state than that for
monoesters (Scheme 2B).33-37 These transition states can be
considered in the context of two limiting extremes. In the
dissociative limit, there is complete bond cleavage to the leaving
group and no bond formation to the nucleophile. In the
associative limit, there is no bond cleavage to the leaving group
and complete bond formation to the nucleophile.33 A transition
state that is close to the dissociative limit is “loose”, while a
transition state that is close to the associative limit is “tight”. A
transition state that is midway between loose and tight is referred
to as “synchronous” herein, as the reaction can be considered
to occur with the extent of bond breaking to the outgoing group
reflecting the extent of bond making to the incoming group such
that the sum of the axial bond orders is approximately one.
The positions of the transition states for the solution reactions
of phosphate monoesters and diesters relative to the limiting
extremes are represented on two-dimensional reaction coordinate
diagrams, or More-O’Ferrall-Jencks diagrams,38,39 in Figure
Phosphate monoester hydrolysis in solution proceeds through
a loose transition state, with nearly complete bond cleavage to
(18) Polanyi, M. Z. Elektrochem. Z. 1921, 27, 142.
(19) Pauling, L. Chem. Eng. News 1946, 24, 1375-1377.
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(31) Hengge, A. C.; Edens, W. A.; Elsing, H. J. Am. Chem. Soc. 1994, 116,
5045-5049.
(32) Thatcher, G. R. J.; Kluger, R. AdV. Phys. Org. Chem. 1989, 25, 99-265.
(33) Hengge, A. C.; Onyido, I. Curr. Org. Chem. 2005, 9, 61-74.
(34) Kirby, A. J.; Younas, M. J. Chem. Soc. B 1970, 1165-1172.
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New York, 1987.
(23) Weiss, P. M.; Cleland, W. W. J. Am. Chem. Soc. 1989, 111, 1928-1929.
(24) Hollfelder, F.; Herschlag, D. Biochemistry 1995, 34, 12255-12264.
(25) O’Brien, P. J.; Herschlag, D. J. Am. Chem. Soc. 1999, 121, 11022-11023.
(26) Holtz, K. M.; Catrina, I. E.; Hengge, A. C.; Kantrowitz, E. R. Biochemistry
2000, 39, 9451-9458.
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(28) Nikolic-Hughes, I.; Rees, D. C.; Herschlag, D. J. Am. Chem. Soc. 2004,
126, 11814-11819.
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AP-Catalyzed Phosphate Mono- and Diester Hydrolysis
A R T I C L E S
Figure 1. Reaction coordinate diagrams for phosphate ester hydrolysis in solution. (A, B) Two-dimensional reaction coordinate diagrams for phosphate
monoester (A) and diester (B) hydrolysis. In both cases, a symmetrical transition state is depicted for simplicity. The transition state can be asymmetrical
for nucleophiles and leaving groups with different pKa values, atom identities, or charges.43 (C, D) Cross sections of the free energy surface along the
tightness coordinate showing the relationship between the solution and enzymatic transition states. In Model 1, AP catalyzes phosphate diester hydrolysis
through a looser transition state than that in solution (C). In Model 2, AP catalyzes phosphate diester hydrolysis through a synchronous transition state
similar to that in solution (D).
1A and B. The reactants and products are located in the lower
left and upper right corners, respectively, and the remaining
corners are the dissociative and associative limits. The two-
dimensional reaction coordinate defines a three-dimensional free
energy surface in which the free energy axis is perpendicular
to the page. The reactants and products are in free energy wells.
The transition state is located at a saddle point on the free energy
surface. It is a maximum along the path from reactants to
products but a minimum along the “tightness coordinate”, the
line connecting the dissociative and associative limits. The
transition state for phosphate monoester hydrolysis is located
close to the dissociative limit (Figure 1A), whereas the transition
state for phosphate diester hydrolysis is synchronous, roughly
midway between the extremes (Figure 1B). In the case of
formation of a stable intermediate, an additional free energy
well could occur in the dissociative or associative corner.
Results from initial experiments using both thio-effects and
linear free energy relationships (LFERs) to characterize the
transition state for AP-catalyzed monoester hydrolysis were
interpreted in terms of a transition state that was tighter than
that for the solution reaction.40-42 The apparent change in the
nature of the transition state was attributed to electrophilic
interactions in the enzyme active site. It was later determined
that the LFER experiments were conducted either in conditions
under which the chemical step was not rate limiting or in the
presence of inhibitory concentrations of inorganic phosphate.27
Furthermore, thio-effects in enzymatic reactions are not as
simply interpreted as those in solution reactions due to effects
(40) Breslow, R.; Katz, I. J. Am. Chem. Soc. 1968, 90, 7376-7377.
(41) Hall, A. D.; Williams, A. Biochemistry 1986, 25, 4784-4790.
(42) Han, R.; Coleman, J. E. Biochemistry 1995, 34, 4238-4245.
(38) More O’Ferrall, R. A. J. Chem. Soc. B 1970, 274-277.
(39) Jencks, W. P. Chem. ReV. 1972, 72, 705-718.
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A R T I C L E S
Zalatan and Herschlag
on binding interactions within the active site that can mask
effects arising from the nature of the transition state.24,44-46
Subsequent experiments provided strong evidence for a loose
enzymatic transition state similar to that in solution and
suggested that electrostatic interactions in the active site do not
alter the nature of the transition state.23-28
Although phosphate diester hydrolysis in solution proceeds
through a synchronous transition state that is tighter than that
for monoesters (Scheme 2), the AP active site is optimized for
phosphate monoester hydrolysis through a loose transition state.
There are two general models for how AP can catalyze
phosphate diester hydrolysis. First, the AP active site might favor
a looser transition state for diester hydrolysis relative to the
reaction in solution. Alternatively, AP could be capable of
recognizing and stabilizing a synchronous diester transition state
similar to that in solution. Parts C and D of Figure 1 depict
these possibilities on cross sections of the free energy surface
along the tightness coordinate. The nature of the transition state
for the enzyme-catalyzed reaction depends both on the specific-
ity of the active site for a particular transition state and the
steepness of the “intrinsic” free energy surface. An active site
that is highly specific for loose transition states would favor a
looser transition state for enzymatic phosphate diester hydrolysis,
while an active site that is capable of stabilizing a tighter
transition state would favor a synchronous enzymatic transition
state. The steepness of the intrinsic free energy surface
determines how energetically costly it is to vary the extent of
bonding in the transition state (Figure 2). If the energy well at
the transition state is shallow, there is little energetic cost to
shifting the nature of the transition state. If the energy well is
steep, there will be a significant energetic cost associated with
shifting the transition state, and with a sufficiently steep energy
well even an active site that is highly specific for loose transition
states would be unable to significantly alter the transition state
for diester hydrolysis relative to that in solution.
Figure 2. The energy required to stabilize an altered transition state and
provide a given amount of catalysis depends on the steepness of the intrinsic
free energy surface. More energetic stabilization is required for a steep
intrinsic free energy surface than for a shallow surface (|∆∆GqSteep| >
|∆∆GqShallow|). The steepness of the intrinsic free energy surface refers to
how energetically costly it is to vary the tightness of the transition state.
The LFER approach requires the use of a series of homolo-
gous compounds with leaving groups of varying pKa. While
this approach is highly effective for characterizing solution
reactions, enzymatic reactions can be complicated by specific
enzyme-substrate interactions. However, the existence of an
open binding site without specific leaving group interactions12
and the use of a large set of homologous compounds has allowed
the applications of LFERs to the AP-catalyzed hydrolysis of
phosphate monoesters,25,27 phosphorothioate monoesters,24,26 and
sulfate monoesters.28 In each case, the LFERs suggested a loose
transition state structure, and these compounds all react via loose
transition states in solution. In the previous work, as with the
experiments described here, only the extent of bonding to the
leaving group in the transition state was measured. The absence
of an observed change in bonding to the leaving group in the
previous and current work suggests, most simply, that bonding
to the nucleophile is also unaffected in the enzymatic transition
state relative to that in solution.
The results described herein suggest that the AP-catalyzed
phosphate diester hydrolysis reaction proceeds through a
synchronous transition state, indistinguishable from diester
hydrolysis in solution. The same bimetallo active site therefore
has the ability to recognize both the loose transition state for
phosphate monoester hydrolysis and the synchronous transition
state for phosphate diester hydrolysis. This flexibility could have
assisted in the evolution of a phosphodiesterase on the AP
scaffold.
Results and Discussion
To characterize the transition state for AP-catalyzed phosphate
diester hydrolysis, we compared enzymatic and nonenzymatic
linear free energy relationships (LFERs) for phosphate diester
hydrolysis. LFERs are a powerful tool for characterization of
transition state structure.22,47 The slope of the line correlating
the log of the rate constant with the pKa of the leaving group
(the Brønsted coefficient, âlg) measures the sensitivity of the
transition state to charge buildup at the position of bond cleavage
and reflects the extent of bond cleavage in the transition state.48
Nonenzymatic Hydrolysis of Methyl Phenyl Phosphate
Diesters. Values of âlg reported in the literature for the solution
reactions of phosphate diesters range from -0.64 to -0.97 and
support the general conclusion that the transition state is roughly
synchronous.35,49-52 These studies, however, employed limited
(48) Although âlg reflects the extent of bonding, it is not a direct measurement
of bond order. Solvent interactions, hydrogen bonding, general acid/general
base catalysis, and metal ion interactions can also affect the observed value
of âlg.
(49) Chin, J.; Banaszczyk, M.; Jubian, V.; Zou, X. J. Am. Chem. Soc. 1989,
111, 186-190.
(50) Williams, N. H.; Cheung, W.; Chin, J. J. Am. Chem. Soc. 1998, 120, 8079-
8087.
(51) Liao, X. M.; Anjaneyulu, P. S. R.; Curley, J. F.; Hsu, M.; Boehringer, M.;
Caruthers, M. H.; Piccirilli, J. A. Biochemistry 2001, 40, 10911-10926.
(52) Ba-Saif, S. A.; Davis, A. M.; Williams, A. J. Org. Chem. 1989, 54, 5483-
5486.
(43) Jencks, W. P. Chem. ReV. 1985, 85, 511-527.
(44) Herschlag, D.; Jencks, W. P. Biochemistry 1990, 29, 5172-5179.
(45) Herschlag, D.; Piccirilli, J. A.; Cech, T. R. Biochemistry 1991, 30, 4844-
4854.
(46) Zhang, Y. L.; Hollfelder, F.; Gordon, S. J.; Chen, L.; Keng, Y. F.; Wu, L.;
Herschlag, D.; Zhang, Z. Y. Biochemistry 1999, 38, 12111-12123.
(47) Williams, A. AdV. Phys. Org. Chem. 1992, 27, 1-55.
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AP-Catalyzed Phosphate Mono- and Diester Hydrolysis
A R T I C L E S
substituted phenyl phosphate diesters56 gives a fractional ef-
fective charge of 0.5, consistent with a synchronous transition
state. For comparison, the fractional effective charge in the loose
transition state for phosphate monoester hydrolysis is 0.9,
estimated from the values of âlg and âeq of -1.23 and -1.35,
respectively.30, 56
Rate constants for phosphate diesters with 4-nitro and 4-cyano
phenyl substituents were not included in the fit used to determine
the value of âlg. There are several examples in the literature of
deviations of compounds with 4-nitro or 4-cyano phenyl
substituents from solution LFERS, presumably due to resonance
effects (see Supporting Information). Furthermore, compounds
with 4-nitro and 4-cyano phenyl substituents are unambiguous
outliers from LFERs for AP-catalyzed hydrolysis reactions.24,26,28
However, inclusion of these points in the LFERs for both
nonenzymatic and enzymatic reactions does not affect the
conclusions drawn from the comparison to the AP-catalyzed
reaction.
Scheme 3. Methyl Phenyl Phosphate Diesters
Table 1. Rate Constants for Enzymatic Methyl Phenyl Phosphate
Diester Hydrolysis at 25 °C, pH 8.0 (kcat/KM) and for the
Nonenzymatic Reaction with Hydroxide at 42 °C (kHO
a
-
)
−
phenyl
phenol
pKa
k
cat/KM
kHO
(M-1 s-
)
1
1
substituent
(M-1 s-
)
4-nitro
4-chloro-3-nitro
4-cyano
7.14
7.78
7.95
8.35
8.63
9.02
9.28
9.38
9.95
9.95
(4.8 ( 0.1) × 10-1
(4.8 ( 0.8) × 10-1
(2.4 ( 0.2) × 10-1
(2.4 ( 0.1) × 10-1
(1.7 ( 0.1) × 10-1
(6.2 ( 0.2) × 10-2
(4.4 ( 1.0) × 10-2
(2.5 ( 1.1) × 10-2
(6.8 ( 0.1) × 10-3
(3.9 ( 0.1) × 10-3
(6.9 ( 0.3) × 10-6
(8.2 ( 0.3) × 10-6
(3.5 ( 0.2) × 10-6
(2.4 ( 0.1) × 10-6
(8.2 ( 0.5) × 10-7
(3.5 ( 0.4) × 10-7
(2.8 ( 0.2) × 10-7
(2.1 ( 0.1) × 10-7
(8.6 ( 1.1) × 10-8
(5.8 ( 0.3) × 10-8
3-nitro
3,4-dichloro
3-chloro
3-fluoro
4-chloro
4-fluoro
parent
Enzymatic Hydrolysis of Methyl Phenyl Phosphate Di-
esters. The products of methyl phenyl phosphate diester
hydrolysis are a substituted phenol and methyl phosphate, but
because AP is a proficient monoesterase, the methyl phosphate
product is rapidly hydrolyzed to inorganic phosphate (Pi) and
methanol. To avoid inhibition by Pi, the AP-catalyzed hydrolysis
rates of a series of phosphate diesters were determined with an
AP variant in which Arg166 was mutated to Ser (Scheme 1B).
Pi binds wild type (wt) AP with an affinity of ∼1 µM, which
creates serious complications for kinetic assays that have
detection limits in this concentration range.27 Pi binds R166S
AP with an affinity of 420 µM,2,59 allowing kinetic assays to
be performed under conditions such that the Pi concentration is
well below inhibitory levels.
a pKa values are from Jencks and Regenstein57 except 4-chloro-3-nitro,
which was calculated from the increments of the 4-chloro and 3-nitro groups,
and 3,4-dichloro.58 The uncertainty is the standard deviation from at least
six measurements.
Mutation of Arg166 to Ser does not alter the reactivity of
AP with phosphate diesters, presumably because the arginine
side chain can readily flip out of the active site to allow
phosphate diester binding.2 Furthermore, Arg166 does not have
a significant effect on LFERs for AP-catalyzed hydrolysis
reactions; the values of âlg for hydrolysis of phosphate and
phosphorothioate monoesters are similar upon mutation of
Arg166.25,26 These properties render R166S AP ideally suited
for studies on the AP-catalyzed phosphate diester hydrolysis
reaction.
The values of kcat/KM for R166S AP-catalyzed hydrolysis of
methyl phenyl phosphate diesters are reported in Table 1. Figure
4 shows the leaving group dependence of log(kcat/KM), which
gives a âlg value of -0.95 ( 0.08. Compounds with 4-nitro
and 4-cyano phenyl leaving groups were omitted from the fit
as discussed below.
Figure 3. Leaving group dependence for attack of hydroxide ion on methyl
phenyl phosphate diesters. Values of kHO (M-1 s-1) are from Table 1. The
-
solid line is a least-squares fit to the data with a slope (âlg) of -0.94 (
0.05. Rate constants for compounds with 4-nitrophenyl and 4-cyano
substituents (open circles) were omitted from the fit based on literature
precedent for the occurrence of outliers due to resonance effects (see
Supporting Information).
sets of compounds, included ortho-substituted compounds, or
included a rate constant for the slow reaction of dimethyl
phosphate that was later determined to be an overestimate.53
We therefore obtained a value of âlg for the nonenzymatic
reaction of a series of methyl phenyl phosphate diesters (Scheme
3) with hydroxide ion for comparison to the enzymatic results
with the same series of compounds.
âlg measures the sensitivity of the transition state to charge
buildup at the leaving group oxygen and can be affected by
interactions with the local environment. Therefore, to compare
the enzymatic value of âlg to that obtained for the solution
hydrolysis reaction, binding interactions with the active site Zn2+
ions must be taken into account. Furthermore, the appropriate
Second-order rate constants for the reaction of hydroxide with
the series of methyl phenyl phosphate diesters are reported in
-
Table 1. Figure 3 shows the dependence of log(kHO ) on leaving
group pKa and gives a value of âlg of -0.94 ( 0.05. The
parameter âlg/âeq represents the fractional effective charge in
the transition state, where âeq is the dependence of log(Keq) on
leaving group pKa.54,55 Using -1.73 as an estimate of âeq for
(55) Williams, A. Acc. Chem. Res. 1984, 17, 425-430.
(56) Bourne, N.; Williams, A. J. Org. Chem. 1984, 49, 1200-1204.
(57) Jencks, W. P.; Regenstein, J. Ionization Constants of Acids and Bases. In
Handbook of Biochemistry and Molecular Biology; Fasman, G. D., Ed.;
CRC: Cleveland, OH, 1976.
(58) Bolton, P. D.; Ellis, J.; Hall, F. M. J. Chem. Soc. B 1970, 1252-1255.
(59) Chaidaroglou, A.; Brezinski, D. J.; Middleton, S. A.; Kantrowitz, E. R.
Biochemistry 1988, 27, 8338-8343.
(53) Williams, N. H.; Wyman, P. Chem. Commun. 2001, 1268-1269.
(54) Leffler, J. E. Science 1953, 117, 340-341.
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A R T I C L E S
Zalatan and Herschlag
diester hydrolysis is closest to that predicted for the synchronous
transition state. There is, however, significant uncertainty in the
obs
lg
predicted values of â , due to assumptions required about the
effects of the interactions between the active site Zn2+ ions and
the transition state (see Methods), and the observed value lies
between the values predicted for synchronous and loose
transition states. For AP-catalyzed phosphate monoester hy-
obs
lg
drolysis, the predicted value of â
is -0.69, smaller in
magnitude than the observed value of -0.85.27 The transition
state for phosphate monoester hydrolysis in solution is close to
the dissociative limit, and it is unlikely that the transition state
for AP-catalyzed hydrolysis is looser than that in solution.
Instead, the predictions probably overestimate the effects that
obs
lg
reduce the magnitude of â (Table 2). These comparisons
suggest that the observed value of -0.95 ( 0.08 for AP-
catalyzed phosphate diester hydrolysis corresponds to an ap-
proximately synchronous transition state, similar to that in
solution. Nevertheless, the predicted values of âlg are based on
the behavior of model compounds in solution, and we do not
know how accurate these models are in describing interactions
in the AP active site. Thus, we cannot estimate uncertainties in
the predicted values of âlg, and a shift to a looser transition
state cannot be excluded solely on the basis of comparisons of
the predicted and observed values of âlg. However, the following
analysis of outliers from LFERs provides strong evidence
supporting a more synchronous transition state for phosphate
diesters than phosphate monoesters.
Figure 4. R166S AP-catalyzed hydrolysis of methyl phenyl phosphate
diesters. Values of kcat/KM (M-1 s-1) are from Table 1. The solid line is a
least-squares fit to the data with a slope (âlg) of -0.95 ( 0.08. Rate constants
for compounds with 4-nitrophenyl and 4-cyano substituents (open circles)
were omitted from the fit based on previous results with phosphorothioate
and sulfate esters in which leaving groups with these substituents were
unambiguous outliers.24,26,28
Table 2. Comparison of Predicted and Observed Values of âlg for
AP-Catalyzed Reactionsa
pred
lg
obs
lg
Predicted Transition State Structure
â
â
Monoester Reactionb
Loose
-0.69
-0.25
0.06
Synchronous
Tight
-0.85 ( 0.13
Compounds with 4-nitro and 4-cyano phenyl leaving groups
are outliers from LFERs in several nonenzymatic reactions (see
Supporting Information) and in AP-catalyzed hydrolysis reac-
tions.24,26,28 In a plot of the log of the rate constant for the AP-
catalyzed reactions of phenyl sulfates versus that for the
corresponding phenyl phosphorothioates, the 4-nitro and 4-cyano
outliers fall on the same correlation line as the other leaving
groups in the series, demonstrating that the magnitudes of the
deviations are the same for both reactions.28 This observation
is consistent with the conclusion that sulfate and phosphorothio-
ate monoesters proceed through similar loose transition states
with extensive charge buildup on the phenolate oxygen. In
contrast, the magnitudes of the deviations of 4-nitro and 4-cyano
phenyl leaving groups from the diester LFER are smaller than
observed for the phosphorothioate or sulfate reactions (Figure
5 and Figure S9 in Supporting Information).
Diester Reactionc
Loose
-1.11
-0.85
-0.43
Synchronous
Tight
-0.95 ( 0.08
a Although similar, the observed values of âlg for the monoester and
diester reactions are not directly comparable. For transition states with
identical axial bonding, predicted values of âlg for the monoester reaction
are smaller in magnitude than those for the diester reaction because of
differences in the values of âeq and in estimates for the strength of the
interaction between the leaving group oxygen and the Zn2+ ion in the
transition state. b Values of âlg for the monoester reaction with wt AP are
from O’Brien and Herschlag, 2002.27 The predicted values take into account
changes in nucleophile strength and interactions with active site Zn2+ ions.
Interactions with Arg166 were not taken into account because mutation of
Arg166 to Ser has no significant effect on the value of âlg.25,26 c The
predicted values for the diester reaction were obtained analogous to those
for the monoester reaction as described in the Methods section.
Two simple models that can account for the smaller deviations
both suggest a transition state for AP-catalyzed diester hydrolysis
that is tighter than that for monoester hydrolysis. Evidence
against alternative models is presented below. In model I,
deviations from LFERs arise if resonance delocalization lags
behind charge buildup (i.e., nonperfect synchronization) such
that the leaving group pKa overestimates the ability of a
substituent to stabilize charge buildup in the transition state.60
The lack of resonance stabilization in the transition state would
be more unfavorable for transition states with increased charge
buildup, leading to larger deviations for reactions with looser
transition states. In model II, deviations arise if resonance
solution reference reaction for comparison to the enzymatic
reaction is one with a nucleophile of comparable strength to
the enzymatic nucleophile. These effects are accounted for as
described in the Methods section and used to predict values of
obs
lg
â
for hypothetical loose, synchronous, and tight enzymatic
transition states based on the known values of âlg for solution
reactions of phosphate monoesters, diesters, and triesters
respectively (Table 2).
The following comparisons suggest that the observed value
of âlg of -0.95 ( 0.08 corresponds to a synchronous transition
state, similar to that for reactions of phosphate diesters in
solution. For a synchronous enzymatic transition state analogous
delocalization affects interactions with the active site Zn2+
.
obs
lg
to diester hydrolysis in solution, the value of â is predicted
These active site interactions are likely to be strong in loose
transition states with extensive charge buildup at the phenolate
oxygen. Resonance delocalization of charge could weaken
to be -0.85. For a loose, monoester-like transition state, the
obs
value of â is predicted to be -1.11, and for a tight, triester-
lg
obs
lg
like transition state, the value of â is predicted to be -0.43.
The observed value of -0.95 ( 0.08 for AP-catalyzed phosphate
(60) Bernasconi, C. F. AdV. Phys. Org. Chem. 1992, 27, 119-238.
9
1298 J. AM. CHEM. SOC. VOL. 128, NO. 4, 2006

AP-Catalyzed Phosphate Mono- and Diester Hydrolysis
A R T I C L E S
position. However, this putative interaction would have been
expected to lead to a similar deviation for methyl phenyl
phosphate diesters as was observed for phenyl phosphorothioates
and sulfates. Finally, Arg166 could play a role in the deviations,
as LFERs for AP-catalyzed phenyl phosphorothioate and phenyl
sulfate hydrolysis reactions were obtained with wt AP, but the
LFER for AP-catalyzed phosphate diester hydrolysis was
obtained with R166S AP. Removal of Arg166 has no significant
effect on the magnitude of the deviations for phenyl phospho-
rothioate hydrolysis,26 rendering this model unlikely. Neverthe-
less, the possibility of a more complex model in which the
absence of Arg166 alters the binding modes for methyl phenyl
phosphate diesters cannot be completely eliminated.
Implications for the Nature of the Transition State for
Enzymatic Phosphoryl Transfer Reactions. There have been
many suggestions that enzymatic phosphoryl transfer reactions
proceed through transition states that are altered from their
solution counterparts.61 These proposals are often based on the
expectation that for loose transition states, the nonbridging
phosphate ester oxygen atoms must donate negative charge to
phosphorus to compensate for the loss of bonding to the
incoming and outgoing groups. For tight transition states,
negative charge is expected to build up on the nonbridging atoms
as bonding to the incoming group increases. Positively charged
functional groups in enzyme active sites would then favor an
increase in negative charge buildup on the nonbridging atoms
in the transition state and thus a tighter transition state. However,
if the free energy well along the tightness coordinate at the
transition state is sufficiently steep, then positively charged
active site functional groups would not provide a sufficient
driving force to significantly alter the transition state. (Figure
2 depicts this effect for an enzymatic transition state that is
looser than the solution transition state; the same considerations
apply for a tighter transition state.) Furthermore, the charge
distribution on the nonbridging atoms in a loose or tight
transition state has not been determined experimentally. Com-
putational studies on metaphosphate monoanions suggest that
charge remains localized on the nonbridging atoms.62,63 Thus,
there may be significant negative charge on the nonbridging
atoms in a loose transition state, and interactions with positively
Figure 5. Deviations from the methyl phenyl phosphate diester LFER are
small. LFERs for wt AP-catalyzed hydrolysis of phenyl phosphorothioates
(0, 9)24 and R166S AP-catalyzed hydrolysis of methyl phenyl phosphate
diesters (O, b) are superimposed. The lines are least-squares fits to the
data (open symbols) with a slope (âlg) of -0.77 ( 0.09 for phenyl
phosphorothioates (solid line) and -0.95 ( 0.08 for phosphate diesters
(dotted line). Rate constants for compounds with 4-nitrophenyl and 4-cyano
substituents (filled symbols) were not included in either fit. These leaving
groups significantly deviate from the phosphorothioate LFER, but show
smaller deviations from the diester LFER.
interactions with the Zn2+, leading to slower reactions than
would be predicted from the leaving group pKa (Figure 6A and
B). In a tighter transition state, there is less total charge buildup
at the phenolate oxygen, which results in a weaker interaction
with the active site Zn2+ and a correspondingly smaller deviation
due to resonance delocalization (Figure 6C and D). Each of the
models described above supports the conclusion that the
transition state for AP-catalyzed phosphate diester hydrolysis
is synchronous.
Alternative models to account for the deviations have been
considered and ruled out as follows. A change in the rate-
determining step can be ruled out on the basis of the substantially
faster hydrolysis rates of 3,4-dinitrophenyl phosphorothioate
(leaving group pKa 5.4) and methyl 2,4-dinitrophenyl phosphate
(leaving group pKa 4.1) relative to the respective 4-nitro
analogues.2,24 A specific, detrimental interaction with the AP
active site could lead to deviations for bulky groups at the para
Figure 6. Resonance effects weaken interactions with active site Zn2+ for a loose transition but have a smaller effect on a tight transition state. (A) There
is a large amount of charge buildup on the bridging oxygen in the transition state for hydrolysis of phenyl phosphorothioates (3-chlorophenyl phosphorothioate
is arbitrarily chosen as an example). (B) 4-Nitro and 4-cyano groups delocalize electron density buildup at the bridging oxygen and weaken the interaction
with the active site Zn12+, leading to a smaller than expected overall rate enhancement. (C) In the tighter transition state for the phosphate diester hydrolysis
reaction, there is less charge buildup at the bridging oxygen. Interactions with Zn12+ may be weaker and therefore less important for the rate enhancement.
2+
(D) 4-Nitro and 4-cyano groups delocalize charge, but if the interaction with Zn1 is weaker, the deviation from the expected rate enhancement will be
smaller.
9
J. AM. CHEM. SOC. VOL. 128, NO. 4, 2006 1299

A R T I C L E S
Zalatan and Herschlag
charged functional groups in enzyme active sites do not
necessarily favor a tighter transition state.
in the transition state and could be caused in part by presence
of the weaker enzymatic nucleophile relative to hydroxide ion
in solution.79 However, the values of âlg for some metal-
substituted variants of phosphotriesterase are significantly more
negative than the value of âeq,78 which in principle represents
the limiting value for full bond cleavage. These results are
difficult to account for and may reflect negative charge buildup
in a hydrophobic active site environment.76
It has also been suggested that the distance between the two
metals ions in the AP active site corresponds to the distance
between the entering and leaving groups in the transition state
and is predictive of the nature of the enzymatic transition state.64
However, the results described herein suggest that AP catalyzes
reactions with phosphate monoesters and diesters through
transition states that have different amounts of bonding to the
axial groups with the same Zn2+-Zn2+ distance. Nevertheless,
it is possible that different Zn2+-Zn2+ distances lead to
preferential catalysis of different reactions. Also, the AP active
site may allow some motion of the bound Zn2+ ions, and
different microstates may be more adept at catalyzing reactions
of different substrates.
A growing body of experimental results from LFER and
kinetic isotope effect (KIE) studies suggests that enzyme-
catalyzed phosphoryl transfer reactions generally proceed
through transition states that are similar to their respective
solution transition states. For example, phosphate monoesters
have been suggested to proceed through loose, solution-like
transition states in the reactions catalyzed by protein tyrosine
phosphatases,65-67 serine/threonine protein phosphatases,68,69
GTPases,70 protein tyrosine kinases,71,72 and acylphosphatases.73
Phosphate diesters have been suggested to proceed through
synchronous transition states in the reactions catalyzed by
kanamycin nucleotidyltransferase74 and the ribonuclease P
ribozyme.75 Diester hydrolysis catalyzed by phosphodiesterase
I was also suggested to proceed through a synchronous transition
state, but with protonation of a nonbridging oxygen based on
the relatively large inverse nonbridging KIEs.37 The results of
subsequent KIE studies of other enzyme-catalyzed phosphoryl
transfer reactions, however, suggest that these results could be
a consequence of active site metal ion interactions rather than
protonation.68
Results from recent structural studies suggest a different
picture may hold for some enzymes. The interconversion of
phosphoenolpyruvate and phosphonopyruvate catalyzed by
phosphoenolpyruvate mutase has been suggested to proceed via
a fully dissociative mechanism with a metaphosphate intermedi-
ate based on the retention of stereochemistry of the reaction
and the apparent lack of an enzymatic nucleophile that would
enable a double displacement mechanism.80 The presence of
metaphosphate was suggested from the crystal structure of
fructose-1,6-bisphosphatase,81 and phosphorane intermediates
were suggested from the crystal structures of â-phosphogluco-
mutase82 and phospholipase D,83 although the â-phosphoglu-
comutase case in particular remains controversial.84-88 These
results have been suggested to support reaction mechanisms that
proceed through formal intermediates instead of a concerted
mechanism through a single transition state. Some enzymatic
glycosyl transfer reactions have been suggested to proceed
through oxycarbenium intermediates, whereas most proceed
through loose, oxycarbenium-like transition states without an
intermediate.89,90 Related solution reactions can also proceed
with or without the formation of a discrete oxycarbenium
intermediate.91,92
The suggestion that the transition states for several enzymatic
phosphoryl transfer reactions are similar to their solution
counterparts is consistent with the expectation that less energy
is required to stabilize a solution-like transition state than a
substantially altered transition state (see Figure 1C,D; |∆∆Gq1|
> |∆∆Gq2|). The data obtained in this work suggest that even
an active site that is optimized for a loose transition state does
not alter the transition state for a reaction that proceeds through
a tighter transition state in solution. Nevertheless, there may be
situations in which the transition state structure diverges
substantially from that observed in solution. The active site
features that might lead to such departures are not yet
understood.
KIE studies of the reaction catalyzed by phosphotriesterase
also suggest a transition state similar to that in solution,76
although there are some distinguishing features of the enzymatic
reaction. The primary KIE for enzymatic O,O-diethyl-O-(4-
carbamoylphenyl) phosphate hydrolysis is larger than that for
alkaline hydrolysis.76 Similarly, values of âlg are more negative
than those for solution reactions.77,78 These results were sug-
gested to reflect increased cleavage of the leaving group bond
Implications for Catalysis and Evolution. AP-catalyzed
phosphate monoester hydrolysis proceeds through a loose
(61) See references in O’Brien and Herschlag, 1999, and Nikolic-Hughes, Rees,
and Herschlag, 2004.25,28
(62) Rajca, A.; Rice, J. E.; Streitwieser, A.; Schaefer, H. F. J. Am. Chem. Soc.
1987, 109, 4189-4192.
(63) Horn, H.; Ahlrichs, R. J. Am. Chem. Soc. 1990, 112, 2121-2124.
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Biochemistry 1991, 30, 7438-7444.
(65) Hengge, A. C. Acc. Chem. Res. 2002, 35, 105-112.
(66) Grzyska, P. K.; Kim, Y.; Jackson, M. D.; Hengge, A. C.; Denu, J. M.
Biochemistry 2004, 43, 8807-8814.
(78) Hong, S. B.; Raushel, F. M. Biochemistry 1996, 35, 10904-10912.
(79) Khan, S. A.; Kirby, A. J. J. Chem. Soc. B 1970, 1172-1182.
(80) Liu, S. J.; Lu, Z. B.; Jia, Y.; Dunaway-Mariano, D.; Herzberg, O.
Biochemistry 2002, 41, 10270-10276.
(67) McCain, D. F.; Grzyska, P. K.; Wu, L.; Hengge, A. C.; Zhang, Z. Y.
Biochemistry 2004, 43, 8256-8264.
(81) Choe, J. Y.; Iancu, C. V.; Fromm, H. J.; Honzatko, R. B. J. Biol. Chem.
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(68) Hengge, A. C.; Martin, B. L. Biochemistry 1997, 36, 10185-10191.
(69) Hoff, R. H.; Mertz, P.; Rusnak, F.; Hengge, A. C. J. Am. Chem. Soc. 1999,
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(71) Kim, K.; Cole, P. A. J. Am. Chem. Soc. 1997, 119, 11096-11097.
(72) Kim, K.; Cole, P. A. J. Am. Chem. Soc. 1998, 120, 6851-6858.
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10547-10559.
(76) Caldwell, S. R.; Raushel, F. M.; Weiss, P. M.; Cleland, W. W. Biochemistry
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9
1300 J. AM. CHEM. SOC. VOL. 128, NO. 4, 2006

AP-Catalyzed Phosphate Mono- and Diester Hydrolysis
A R T I C L E S
transition state with a >1017-fold rate enhancement, while
phosphate diester hydrolysis proceeds through a tighter, syn-
chronous transition state with a 1011-fold rate enhancement.2
The smaller rate enhancement for diester hydrolysis indicates
that the AP active site binds the synchronous transition state
for diester hydrolysis transition state more weakly than it binds
the loose transition state for monoester hydrolysis and suggests
that the looseness or tightness of the transition state could
account for the different rate enhancements. Other features of
the transition state, however, must also be important for
determining the rate enhancement for AP-catalyzed reactions.
AP-catalyzed sulfate monoester hydrolysis proceeds through a
loose transition state similar to phosphate monoester hydroly-
sis,28 but with a rate enhancement of only 109-fold.93
catalyzed by AP already proceeds through a synchronous
transition state. Optimization of this activity then simply requires
improved binding of the synchronous transition state (increasing
|∆∆Gq2| in Figure 1D).
Conclusions and Future Directions
The results described herein suggest that AP catalyzes
phosphate diester hydrolysis through a synchronous transition
state that is indistinguishable from that in solution, even though
the AP active site is optimized for the loose transition state of
the phosphate monoester hydrolysis reaction. Thus, evolution
of a proficient phosphodiesterase on the AP-scaffold could occur
without the need to alter the transition state structure. It will be
fascinating to compare the AP active site to homologous active
sites that preferentially catalyze phosphate diester hydrolysis.
Determining the physical changes that have occurred in the AP
active site to optimize phosphodiesterase activity will improve
our understanding of the catalytic mechanisms of this important
class of enzymes, provide insight into how new enzyme
activities evolve, and assist in the engineering of artificial
enzymes.
Additional active site interactions can contribute to discrimi-
nation between phosphate monoester and diester substrates.
Mutation of Arg166 to Ser leads to a 104-fold decrease in kcat/
K
M for phosphate monoesters25 but does not affect the rates of
phosphate diester reactions,2 suggesting that this residue is
important for binding and positioning of monoester substrates.
To optimize diesterase activity on the AP scaffold, binding
interactions with the diester functionality on the transferred
phosphoryl group (R′ in Scheme 1B) could be introduced to
assist with specific binding and positioning of diester substrates.
Materials and Methods
Materials. The plasmid for expression of R166S AP (pEK1152)
and the phoA- strain of E. coli (SM547) were provided by Evan
Kantrowitz.59 HPLC grade acetone was used for synthesis without
distillation.
Synthesis of Methyl Phenyl Phosphate Diesters. Lithium salts of
methyl phenyl phosphate diesters where prepared as described52 except
that the reflux step was extended to 2 h and the product was purified
on silica gel using ethyl acetate/hexanes/methanol in a gradient from
(20:80:0) to (20:60:20) instead of recrystallization. Yields averaged
∼25%. Purified products were characterized by 1H and 31P NMR on a
Varian Mercury 400 MHz instrument in D2O solvent. NMR chemical
shift data are reported in Supporting Information.
Nonenzymatic Phosphate Diester Hydrolysis Kinetics. Reactions
of hydroxide ion with phosphate diesters were performed at 42 °C,
and ionic strength was adjusted to 1.0 with NaCl. Absorbance of the
phenolate ion product was monitored continuously using a Uvikon 9310
spectrophotometer at the following wavelengths: 4-nitro (400 nm),
4-chloro-3-nitro (400 nm), 4-cyano (274 nm), 3-nitro (390 nm), 3,4-
dichloro (302 nm), 3-chloro (291 nm), 3-fluoro (282 nm), 4-chloro (296
nm), 4-fluoro (297 nm), and parent (286 nm). Bimolecular rate constants
were obtained from initial rates (e2% reaction). Reactions were shown
to be first order in hydroxide ion from 0.05 to 0.5 M and in phosphate
diester over a 10-fold concentration range (between the limits of 0.1
and 10 mM). The first-order dependence on hydroxide ion concentration
demonstrates that the phosphate diester monoanion is the reactive
species. Under these conditions, direct attack by hydroxide at phos-
phorus is the dominant reaction.95 Two alternative reaction possibilities
can be ruled out as follows. First, isotope incorporation studies rule
out nucleophilic aromatic substitution by attack at the phenolic carbon
of the leaving group.36 Second, hydroxide attack at the methyl carbon
followed by subsequent hydrolysis of a phosphate monoester can be
ruled out because phosphate monoester hydrolysis, which would be
required to give the phenolate and the observed increase in absorbance,
is too slow to account for the observed reaction rates.30,96
Electrostatic properties of the nonbridging phosphate ester
oxygen atoms and their interactions with the bimetallo center
have also been suggested to be important. The rate enhance-
ments for R166S AP-catalyzed hydrolysis reactions correlates
with the amount of negative charge situated between the two
Zn2+ ions in the AP active site, suggesting that these electrostatic
interactions make a large contribution to catalysis.94 Neverthe-
less, there are many examples of binuclear metalloenzymes that
preferentially hydrolyze phosphate diesters.16,17 Thus, the pres-
ence of two closely spaced divalent cations does not necessarily
lead to preferential monoester hydrolysis. As noted above,
diester hydrolysis could be favored by binding interactions with
the transferred substituent that is absent on a monoester substrate
(R′ in Scheme 1B). However, it is also possible that active site
features tune the electrostatic properties of the bimetallo cluster
by varying the coordinating ligands, the spacing between the
metal ions, or the presence or absence of other nearby charged
groups to favor reactions of monoesters or diesters.
We now consider evolutionary scenarios in which a diesterase
evolved from AP; the same considerations apply if AP were to
have evolved from a diesterase. If AP-catalyzed phosphate
diester hydrolysis proceeded through a loose, monoester-like
transition state, then evolution of a diesterase might have
involved shifting the transition state back to a synchronous,
solution-like transition state. Such a change could have played
an important role in optimization of catalysis, as less energy is
required to stabilize a solution-like transition state than a
substantially altered transition state. Alternatively, a scenario
in which other mechanisms were employed to provide greater
stabilization of a looser transition state for the diesterase reaction
(increasing |∆∆Gq1| in Figure 1C) might have been possible.
These evolutionary scenarios, however, can be ruled out by the
observation that the phosphate diester hydrolysis reaction
Alkaline Phosphatase Purification. R166S alkaline phosphatase
was purified as previously described,2 except that the dialysis step was
omitted. Protein concentration was determined by absorbance at 280
(95) Cassano, A. G.; Anderson, V. E.; Harris, M. E. J. Am. Chem. Soc. 2002,
124, 10964-10965.
(96) Lad, C.; Williams, N. H.; Wolfenden, R. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 5607-5610.
(93) O’Brien, P. J.; Herschlag, D. J. Am. Chem. Soc. 1998, 120, 12369-12370.
(94) Nikolic-Hughes, I.; O’Brien, P. J.; Herschlag, D. J. Am. Chem. Soc. 2005,
127, 9314-9315.
9
J. AM. CHEM. SOC. VOL. 128, NO. 4, 2006 1301

A R T I C L E S
Zalatan and Herschlag
Scheme 4. Binding Interactions Affect âlg
from this work of -0.94 (see Results) gives âlg,nonenz ) -0.94 + -0.17
) -1.11 (eq 2).
âqbind (Scheme 4) has contributions from interactions with the active
site Zn2+ at both the bridging and nonbridging phosphate ester oxygen
atoms in the transition state (Scheme 1 and eq 3). The bridging
contribution can be estimated from the literature value99 of âZphnebnionldate
)
0.36 and the fractional effective charge in the transition state54,55
(âlg/âeq) according to:
âqbridge Zn bind ) âphenolate × (fractional effective charge) (4)
Zn bind
Using âlg,nonenz ) -1.11 and âeq ) -1.73,56 the fractional effective
charge is âlg,nonenz/âeq ) 0.64, giving âqbridge Zn bind ) 0.36 × 0.64 )
0.23 (eq 4). âqnonbridge Zn bind accounts for the change in Zn2+ affinity to
the nonbridging oxygen atom of a phosphate diester in the transition
state with the pKa of the leaving group according to:
âqnonbridge Zn bind pKROH
(5)
nonbridge
Zn
nm (ꢀ280 ) 3.14 × 104 M-1 cm-1 in monomer units).97 Concentrations
determined by activity assays using 1 mM 4-nitrophenyl phosphate
agreed to within 20% (kcat ) 0.44 s-1).59
log (K
)
This quantity cannot be directly measured, but it can be crudely
approximated from literature data. Equation 6 correlates the binding
constants of phosphate esters to Zn2+ with the pKa of the phosphate
Enzymatic Phosphate Diester Hydrolysis Kinetics. Reactions were
performed with 0.1 M NaMOPS pH 8.0, 0.5 M NaCl, 1 mM MgCl2,
and 0.1 mM ZnSO4 at 25 °C. Appearance of phenolate was monitored
by removing aliquots from the reaction, quenching in an equal volume
of 0.1 M NaOH, and measuring the absorbance using a Uvikon 9310
at the wavelengths listed above. Rate constants were obtained from
initial rates (e2% reaction). Reactions were shown to be first order in
substrate and enzyme by varying substrate concentration over a 10-
fold range (between the limits of 0.1 and 10 mM) and enzyme
concentration from 1 to 10 µM. Apparent second-order rate constants
kcat/KM are reported per active site. For 4-nitro, 4-chloro-3-nitro, 4-cyano,
3-nitro, and 3,4-dichloro substrates, rates constants were also determined
independently by monitoring phenolate absorbance continuously at pH
8.0, and the rate constants agreed with those from the quench assay to
within 2% in side-by-side comparisons.
ester (pKROP).100 eq 7 correlates the pKa of phosphate diesters (pKROP
)
with the pKa of the leaving group (pKROH),56 which can be used to
convert a dependence on pKROP to a dependence on pKROH. Combining
eqs 6 and 7 gives an estimate for âqnonbridge Zn bind of 0.03 (eq 8). Then
âbqind ) âqbridge Zn bind + ânqonbridge Zn bind ) 0.23 + 0.03 ) 0.26 (eq 3).
nonbridge
Zn
log(K
)
0.35 pKROP
(6)
(7)
pKROP 0.09 pKROH
nonbridge
Zn
log(K
)
0.03 pKROH
(8)
The contributions from binding interactions and changing nucleophile
strength predict âobs ) âlg,nonenz + âq ) -1.11 + 0.26 ) -0.85 (eq
lg
bind
To confirm that the observed diesterase activity was due to R166S
AP and not contamination by a small quantity of a proficient diesterase,
the inhibition constant for Pi was measured with 4-nitrophenyl
phosphate, a phosphate monoester, and several phosphate diester
substrates. The values of the inhibition constant for the monoester and
diester reactions agreed to within 10% (data not shown), strongly
suggesting that the monoesterase and diesterase reactions occurred in
the same active site in R166S AP.2
1) for an enzymatic transition state similar to that for phosphate diester
hydrolysis in solution.
Comparison for a Monoester-Like Transition State. For phosphate
monoester hydrolysis, âlg ) -1.23.30 Accounting for nucleophile
strength from neutral water to Ser-O-‚‚‚Zn2+ using pxy ) 0.013101 gives
∆âlg,nonenz ) pxy × ∆pKnuc ) 0.013 × (7 - (-1.7)) ) 0.11 and
monoester
lg,nonenz
monoester
) -1.35,56
eq
â
) -1.23 + 0.11 ) -1.12 (eq 2). From â
the fractional effective charge is âmlgo,nnooneesntezr/âmonoester ) 0.83. If the
eq
Comparison of AP-Catalyzed and Nonenzymatic Values of âlg
for a Diester-Like Transition State. Comparison of âlg between
solution and enzymatic reactions was undertaken following a previously
described approach for AP-catalyzed reactions.27 Binding interactions
with the active site Zn2+ (Scheme 4) and changing the nucleophile
strength are expected to affect the observed value of âlg according to:
diester were to proceed through a transition state with similar fractional
diester
effective charge, âdiester
lg,nonenz/â
would equal 0.83. Given the value of
eq
diester
eq
â
) -1.73, âdiester
lg,nonenz would equal -1.44. For binding interactions:
âqbridge Zn bind ) âphenolate × (fractional effective charge) ) 0.36 × 0.83
Zn bind
) 0.30 (eq 4) and âqnonbridge Zn bind ) 0.03 as described above for a
diester-like transition state, assuming that there would not be drastic
changes in the charge distribution at the nonbridging oxygen atoms.
This term is small and unlikely to influence the outcome of this analysis.
obs
lg
â
) âlg,nonenz + âbqind
(1)
(2)
It then follows that âqbind ) 0.30 + 0.03 ) 0.33 (eq 3) and âobs
)
lg
âlg,nonenz ) âlg, OH + pxy∆pKnuc
-
diester
â
+ âqbind ) -1.44 + 0.33 ) -1.11 (eq 1) for an enzymatic
lg,nonenz
âbqind ) âqbridge Zn bind + âqnonbridge Zn bind
(3)
transition state similar to that for phosphate monoester hydrolysis in
solution.
Comparison for a Triester-Like Transition State. For phosphate
triester hydrolysis, âlg ) -0.99,79 accounting for nucleophile strength
from neutral water to Ser-O-‚‚‚Zn2+ using pxy ) 0.04479 gives
∆âlg,nonenz ) pxy × ∆pKnuc ) 0.044 × (7 - (-1.7)) ) 0.38 and
The interaction coefficient pxy describes the change in âlg with
changing nucleophile strength (∆pKnuc) relative to a reference reaction
(eq 2). The pKa of the Ser-O-‚‚‚Zn2+ nucleophile in the E‚S complex
is estimated to be ∼7.98 Relative to a solution reaction with hydroxide
ion (pKa ) 15.7) as the nucleophile and using pxy ) 0.019,35 Ɖlg,nonenz
(99) Postmus, C.; Magnusson, L. B.; Craig, C. A. Inorg. Chem. 1966, 5, 1154-
1157.
-
) pxy × ∆pKnuc ) 0.019 × (7-15.7) ) -0.17. The value of âlg,
OH
(100) Massoud, S. S.; Sigel, H. Inorg. Chem. 1988, 27, 1447-1453. The
correlation of binding constants of phosphate esters to Zn2+ with the pKa
of the phosphate ester (eq 5) was determined for phosphate monoesters.
We assume that this correlation also holds for diesters.
(101) Herschlag, D.; Jencks, W. P. J. Am. Chem. Soc. 1989, 111, 7587-7596.
(102) Baes, C. F., Jr.; Mesmer, R. E. The Hydrolysis of Cations; Wiley: New
York, 1976.
(97) Gill, S. C.; von Hippel, P. H. Anal. Biochem. 1989, 182, 319-326.
(98) The pKa of Ser-O-‚‚‚Zn2+ in the E‚S complex has not been experimentally
determined. The pKa in the free enzyme is e5.5,27 and binding of a
negatively charged substrate is expected to increase the pKa. Using a pKa
of 4 or of 9, the pKa of a Zn2+-bound water,102 does not significantly alter
the results of this analysis and does not affect the conclusions drawn.
9
1302 J. AM. CHEM. SOC. VOL. 128, NO. 4, 2006

AP-Catalyzed Phosphate Mono- and Diester Hydrolysis
A R T I C L E S
triester
lg,nonenz
triester
eq
â
) -0.99 + 0.38 ) -0.61 (eq 2). From â
) -1.82,56 the
) 0.34. If the diester
were to proceed through a transition state with similar fractional
Acknowledgment. This work was supported by a grant from
the NIH to D.H. (GM64798). J.G.Z. was supported in part by
a Hertz Foundation Graduate Fellowship. We thank members
of the Herschlag lab for comments on the manuscript.
fractional effective charge is âtriester
lg,nonenz/â
triester
eq
effective charge, âdiester
lg,nonenz/â
would equal 0.34. Given the value of
diester
eq
diester
eq
â
) -1.73, âdiester
lg,nonenz would equal -0.59. For binding interactions:
Supporting Information Available: Data compiled from the
literature for nonenzymatic LFERS with 4-nitro and 4-cyano
phenyl leaving groups, plots of the log of the rate constant for
the AP-catalyzed reactions of phosphate diesters and phenyl
sulfates versus that for the corresponding phenyl phosphorothio-
ates, and NMR chemical shift data for methyl phenyl phosphate
diesters. This material is available free of charge via the Internet
at http://pubs.acs.org.
âqbridge Zn bind ) âphenolate × (fractional effective charge) ) 0.36 × 0.34
Zn bind
) 0.12 (eq 4) and âqnonbridge Zn bind ) 0.03 as described above for a
diester-like transition state, assuming that there would not be drastic
changes in the charge distribution at the nonbridging oxygen atoms.
This term is small and unlikely to influence the outcome of this analysis.
It then follows that âqbind ) 0.12 + 0.03 ) 0.15 (eq 3) and âobs
)
lg
triester
lg,nonenz
â
+ âqbind ) -0.58 + 0.15 ) -0.43 (eq 1) for an enzymatic
transition state similar to that for phosphate triester hydrolysis in
solution.
JA056528R
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J. AM. CHEM. SOC. VOL. 128, NO. 4, 2006 1303