Do electrostatic interactions with positively charged active site groups tighten the transition state for enzymatic phosphoryl transfer?

Article abstract of DOI:10.1021/ja0480421

The effect of electrostatic interactions on the transition-state character for enzymatic phosphoryl transfer has been a subject of much debate. In this work, we investigate the transition state for alkaline phosphatase (AP) using linear free-energy relationships (LFERs). We determined f_cat/K _M for a series of aryl sulfate ester monoanions to obtain the Bronsted coefficient, β_Ig, and compared the value to that obtained previously for a series of aryl phosphorothioate ester dianion substrates. Despite the difference in substrate charge, the observed Bronsted coefficients for AP-catalyzed aryl sulfate and aryl phosphorothioate hydrolysis (-0.76 ± 0.14 and -0.77 ± 0.10, respectively) are strikingly similar, with steric effects being responsible for the uncertainties in these values. Aryl sulfates and aryl phosphates react via similar loose transition states in solution. These observations suggest an apparent equivalency of the transition states for phosphorothioate and sulfate hydrolysis reactions at the AP active site and, thus, negligible effects of active site electrostatic interactions on charge distribution in the transition state.

Full text of DOI:10.1021/ja0480421

Published on Web 09/01/2004
Do Electrostatic Interactions with Positively Charged Active
Site Groups Tighten the Transition State for Enzymatic
Phosphoryl Transfer?
Ivana Nikolic-Hughes,†,‡ Douglas C. Rees,‡,§ and Daniel Herschlag*,†,|,
Contribution from the Departments of Chemical Engineering, Chemistry, and Biochemistry,
Stanford UniVersity, Stanford, California 94305, DiVision of Chemistry and Chemical
Engineering and Howard Hughes Medical Institute, California Institute of Technology,
Pasadena, California 91125
Received April 5, 2004; E-mail: herschla@cmgm.stanford.edu
Abstract: The effect of electrostatic interactions on the transition-state character for enzymatic phosphoryl
transfer has been a subject of much debate. In this work, we investigate the transition state for alkaline
phosphatase (AP) using linear free-energy relationships (LFERs). We determined kcat/KM for a series of
aryl sulfate ester monoanions to obtain the Brønsted coefficient, âlg, and compared the value to that obtained
previously for a series of aryl phosphorothioate ester dianion substrates. Despite the difference in substrate
charge, the observed Brønsted coefficients for AP-catalyzed aryl sulfate and aryl phosphorothioate hydrolysis
(-0.76 ( 0.14 and -0.77 ( 0.10, respectively) are strikingly similar, with steric effects being responsible
for the uncertainties in these values. Aryl sulfates and aryl phosphates react via similar loose transition
states in solution. These observations suggest an apparent equivalency of the transition states for
phosphorothioate and sulfate hydrolysis reactions at the AP active site and, thus, negligible effects of active
site electrostatic interactions on charge distribution in the transition state.
Introduction
has been proposed numerous times that electrostatic interactions
between these positive charges and the phosphoryl group would
Catalysis is defined as preferential stabilization of a reaction’s
transition state, relative to its ground state.1 Thus, detailed
knowledge of the transition-state character for both nonenzy-
matic and enzymatic reactions is essential to decipher enzymatic
catalysis. The least amount of energy is required for an enzyme
that stabilizes a transition state closely related to that found in
solution, and many enzymatic transition states have indeed been
found to be similar to their nonenzymatic counterparts.2
Physical organic studies over the past several decades have
characterized the nonenzymatic transition state for phosphoryl
transfer from phosphate monoesters as loose, with a large
amount of bond breaking to the leaving group and only a small
amount of bond formation to the nucleophile, presumably
resulting in a reduction of negative charge on the nonbridging
oxygen atoms of the phosphoryl group, relative to the ground
state (eq 1).3 As enzymes tend to surround the phosphoryl group
with hydrogen bond donors and positively charged groups, it
introduce an energetic incentive to increase the amount of charge
on the nonbridging oxygen atoms in the transition state, thereby
tightening the transition state, such that there is less bond
breaking to the leaving group and more bond formation to the
nucleophile (Scheme 1).4
Recently, Escherichia coli alkaline phosphatase (AP) has been
shown to have activity toward sulfate monoesters in addition
to the cognate phosphate monoester substrates.5 The AP active
site contains two Zn2+ metal ions and an arginine residue that
interact directly with the transition state (Scheme 2).6 Because
(3) For review, see: (a) Thatcher, G. R. J.; Kluger, R. AdV. Phys. Org. Chem.
1989, 25, 99. (b) Hengge, A. C. In ComprehensiVe Biological Catalysis;
Sinnott, M. L., Ed.; Academic Press: London, 1998; Vol. 1, pp 517-542.
(4) For example, see: (a) Breslow, R.; Katz, I. J. Am. Chem. Soc. 1968, 90,
7370. (b) Hall, A. D.; Williams, A. Biochemistry 1986, 25, 4784. (c)
Mildvan, A. S.; Fry, D. C. AdV. Enzymol. 1987, 59, 241. (d) Reinstein, J.;
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meyer, D.; Engh, R. A.; Kinzel, V.; Ponstingl, H.; Huber, R. EMBO J.
1993, 12, 849. (f) Abrahams, J. P.; Leslie, A. G. W.; Lutter, R.; Walker,
J. E. Nature 1994, 370, 621. (g) Goldberg, J.; Huang, H.; Kwon, Y.;
Greengard, P.; Nairn, A. C.; Kuriyan, J. Nature 1995, 376, 745. (h)
Berghuis, A. M.; Lee, E.; Raw, A. S.; Gilman, A. G.; Sprang, S. R. Structure
1996, 4, 1277. (i) Rittinger, K.; Walker, P. A.; Eccleston, J. F.; Smerdon,
S. J.; Gamblin, S. J. Nature 1997, 389, 758. (j) Matte, A.; Tari, L. W.;
Delbaere, L. T. J. Structure 1998, 6, 413. (k) Schlichting, I.; Reinstein, J.
Nat. Struct. Biol. 1999, 6, 721. (l) Singh-Wissmann, K.; Miles, R. D.;
Ingram-Smith, C.; Ferry, J. G. Biochemistry 2000, 39, 3671. (m) Madhusudan,
A. P.; Xuong, N.-H.; Taylor, S. S. Nat. Struct. Biol. 2002, 9, 273. (n)
Delbaere, L. T. J.; Sudom, A. M.; Prasad, L.; Leduc, Y.; Goldie, H. Biochim.
Biophys. Acta 2004, 1697, 271. See also: (o) Glennon, T. M.; Warshel, A.
Biochemistry 2000, 39, 9641. (p) Hutter, M. C.; Helms, V. Int. J. Quantum
Chem. 2003, 95, 479. (q) Cai, M.; Williams, D. C., Jr.; Wang, G.; Lee, B.
R.; Peterkofsky, A.; Clore, G. M. J. Biol. Chem. 2003, 278, 25191. (r)
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(5) O’Brien, P. J.; Herschlag, D. J. Am. Chem. Soc. 1998, 120, 12369.
† Department of Chemical Engineering, Stanford University.
‡ Division of Chemistry and Chemical Engineering, California Institute
of Technology.
§ Howard Hughes Medical Institute.
| Department of Biochemistry, Stanford University.
Department of Chemistry, Stanford University.
(1) (a) Polanyi, M. Z. Elektrochem. Z. 1921, 27, 142. (b) Pauling, L. Chem.
Eng. News 1946, 24, 1375. (c) Jencks, W. P. In Catalysis in Chemistry
and Enzymology; McGraw-Hill: New York, 1969.
(2) For example, see: (a) Kempton, J. B.; Withers, S. G. Biochemistry 1992,
34, 9961. (b) Bruner, M.; Horenstein, B. A. Biochemistry 1998, 37, 289.
(c) Scheuring, J.; Schramm, V. L. Biochemistry 1997, 36, 4526. (d) Berti,
P. J.; Schramm, V. L. J. Am. Chem. Soc. 1997, 119, 12069. See also: (e)
Yang, J.; Schenkman, S.; Horenstein, B. A. Biochemistry 2000, 39, 5902.
Reviewed in: (f) Schramm, V. L. Curr. Opin. Chem. Biol. 2001, 5, 556.
9
11814
J. AM. CHEM. SOC. 2004, 126, 11814-11819
10.1021/ja0480421 CCC: $27.50 © 2004 American Chemical Society

Interactions with Positive Active Site Groups
A R T I C L E S
Scheme 1. Observed and Hypothetical Transition States for
Phosphoryl Transfer
Scheme 2. AP Active Site with a Bound Transition-State Model6
of the AP active site with phosphate monoester dianions result
in a tighter transition state, whereas sulfate ester monoanions
undergo a smaller change (if any) because their lower charge
results in less intimate, more distant electrostatic interactions
(Scheme 3A). (2) The lower charge density of the sulfate ester
monoanions leads to an imbalance of charge, especially on the
nonbridging oxygen that interacts with both active site Zn2+
ions. This charge imbalance creates a large driving force for
increasing the charge on this nonbridging oxygen atom in the
sulfate ester transition state, whereas the corresponding non-
bridging oxygen of the phosphate ester transition state is more
highly charged so that there is less of a driving force from
interaction with the two Zn2+ ions for a charge increase on this
atom. (Scheme 3B depicts an increase in charge for the sulfuryl
transfer transition state for all of the nonbridging oxygen atoms
relative to the unperturbed transition state (Scheme 3A,C); an
increase in charge in the sulfuryl transfer reaction would most
simply be accompanied by increasing the bond order to the
incoming and outgoing groups, as is also depicted in Scheme
3B.) (3) Bonding in both the phosphoryl and sulfuryl transfer
transition states is unchanged relative to solution because the
“force” exerted by the active site is insufficient to substantially
reorganize the covalent and partially covalent bonds in the
transition state (Scheme 3C).
Additionally, there is literature precedent to suggest that if
the active site environment indeed affected the transition-state
nature, the response of the phosphate ester would be different
from that of the sulfate ester. The effect of dimethyl sulfoxide
(DMSO) addition on nonenzymatic rate constants has been
found to be drastically different for a phosphate ester compared
to a sulfate ester (106 vs 50-fold rate enhancement in 95%
DMSO, respectively).11 This solvent effect presumably arises
from a loss of stabilizing ground-state electrostatic interactions
between substrate and water when the transition state is reached,
as charge is dispersed in the transition state.11c Therefore,
removing electrostatic interactions (via DMSO addition) is
energetically more unfavorable for phosphate than for sulfate
the phosphate monoesters are dianionic, whereas sulfate mono-
esters are monoanionic (eqs 1 and 2), we considered whether
the overall charge difference between these substrates would
result in them being “handled” differently within the highly
charged AP active site (Scheme 2). Although the P-O and S-O
bonds are very similar in length,7 AP catalyzes the phosphate
ester reaction with a catalytic proficiency that is at least 1010-
fold greater than the catalytic proficiency for the sulfate ester
reaction, corresponding to 13 kcal/mol of additional transition-
state stabilization for the phosphate compared to the sulfate.5,8
Additionally, physical organic studies of nonenzymatic sulfate
monoester monoanion hydrolysis suggest a transition state that
is loose, akin to that for the phosphate monoester reactions (eqs
1 and 2).9,10 These and additional observations (O’Brien, P. J.;
Nikolic-Hughes, I.; Herschlag, D, unpublished results) support
the notion that charge is of utmost importance to AP catalysis.
Given the proposals for creation of a tighter transition state
within the highly charged active site of phosphoryl transfer
enzymes, including AP, and the apparent importance of
electrostatics in AP catalysis, we were interested in comparing
the transition states for AP-catalyzed phosphoryl and sulfuryl
transfer. Three classes of models were considered and are
depicted in Scheme 3. (1) The strong electrostatic interactions
(6) Holtz, K. M.; Stec, B.; Kantrowitz, E. R. J. Biol. Chem. 1999, 274, 8351.
(7) CRC Handbook of Chemistry and Physics, 75th ed.; Lide, D. R., Ed.; CRC
Press: Boca Raton, FL, 1994-1995; pp 9-12.
(8) Lad, C.; Williams, N. H.; Wolfenden, R. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 5607.
(9) See: (a) Kirby, A. J.; Jencks, W. P. J. Am. Chem. Soc. 1965, 87, 3209. (b)
Kirby, A. J.; Varvoglis, A. G. J. Am. Chem. Soc. 1967, 89, 415. (c)
Benkovic, S. J.; Benkovic, P. A. J. Am. Chem. Soc. 1966, 88, 5504. (d)
Hengge, A. C. Acc. Chem. Res. 2002, 35, 105. (e) The nonenzymatic
hydrolysis of para-nitrophenyl phosphate and para-nitrophenyl sulfate was
found to occur via P-O and S-O bond cleavage, respectively (ref 9a and
c).
(11) (a) Abell, K. W. Y.; Kirby, A. J. Tetrahedron Lett. 1986, 27, 1085. (b)
Hoff, R. H.; Larsen, P.; Hengge, A. C J. Am. Chem. Soc. 2001, 123, 9338.
(c) Cheng, H.; Nikolic-Hughes, I.; Wang, J. H.; Deng, H.; O’Brien, P. J.;
Wu, L.; Zhang, Z.-Y.; Herschlag, D.; Callender, R. J. Am. Chem. Soc. 2002,
124, 11295.
(10) Fendler, E. J.; Fendler, J. H. J. Org. Chem. 1968, 33, 3852.
9
J. AM. CHEM. SOC. VOL. 126, NO. 38, 2004 11815

A R T I C L E S
Nikolic-Hughes et al.
Scheme 3. Possible Responses of Phosphate and Sulfate Transition States to Electrostatic Interactions within the AP Active Site
(7.8 kcal/mol vs 2.2 kcal/mol, respectively). This suggests that
loss of electrostatic interactions with positively charged active
site groups in the transition state would be more unfavorable
for phosphate than sulfate, and thus a greater force to optimize
those interactions, if indeed such a force existed, would be
exerted upon a phosphate, as described in model 1 (Scheme
3A).
rather than the chemical step,12 whereas thio-substitution renders
the chemical step rate-limiting.13,14
Figure 1 shows a comparison of the leaving group dependence
for the aryl sulfate and aryl phosphorothioate nonenzymatic
hydrolysis, from literature data and aryl sulfate data obtained
in this work (Table 1).10,13 These correlations give the same
slope, within error, for aryl sulfates and aryl phosphorothioates,
with âlg values of -1.2. The results are consistent with the
previous conclusions that these reactions proceed through
similar, loose transition states in aqueous solution.9
Results and Discussion
To determine the effect of electrostatic interactions in the
highly charged active site of AP (Scheme 3 and Introduction)
we compared linear free-energy relationships (LFERs) for
phosphoryl and sulfuryl transfer in solution and in the AP active
site. A series of aryl leaving groups was used to determine the
LFER coefficient âlg, which represents the dependence of the
logarithm of the reaction rate constant on the leaving group pKa.
Phosphorothioates were used in the comparison because the AP-
catalyzed hydrolysis of aryl phosphates is limited by binding
We next determined a Brønsted correlation for AP-catalyzed
hydrolysis of aryl sulfates, using a broader series of synthesized
aryl sulfate esters, and compared this LFER to that previously
(12) (a) Simopoulos, T. T.; Jencks, W. P. Biochemistry 1994, 33, 10375. (b)
Hengge, A. C.; Edens, W. A.; Elsing, H. J. Am. Chem. Soc. 1994, 116,
5045.
(13) Hollfelder, F.; Herschlag, D. Biochemistry 1995, 34, 12255.
(14) Holtz, K. M.; Catrina, I. E.; Hengge, A. C.; Kantrowitz, E. R. Biochemistry
2000, 39, 9451.
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Interactions with Positive Active Site Groups
A R T I C L E S
Figure 2. AP-catalyzed hydrolysis of aryl phosphorothioates (9)13 and
aryl sulfates (b). Aryl sulfates were synthesized,10 and AP was purified as
described previously.5 Aryl sulfate AP-catalyzed hydrolysis reactions were
performed in 0.1 M CHES, pH 10.0, 0.5 M NaCl at 60 °C (see Methods
section). The sulfate series phenol leaving groups and their pKa values are
as follows: 4-nitro (7.14), 4-chloro, 3-nitro (7.78), 4-cyano (7.95), 3-nitro
(8.35), 3,4-dichloro (8.55), 3-chloro (9.02), 3-bromo (9.11), 3-fluoro (9.29),
4-bromo (9.37), 4-chloro (9.38), 4-fluoro (9.95), parent (9.95), and
4-methoxy (10.21). The solid lines represent the best least-squares fit to
the data, with a slope (âlg) of -0.76 ( 0.14 for sulfates and -0.77 ( 0.10
for phosphorothioates. Rate constants for 4-nitrophenyl and 4-cyanophenyl
substituents are represented by open symbols in both series and were not
included in the linear fits.13
Figure 1. Leaving group dependence for nonenzymatic hydrolysis of aryl
sulfates (9, [) and aryl phosphorothioates (O). In the current work, aryl
sulfate esters were synthesized as described previously,10 and their reactions
(9) were conducted at 90 °C, as described in the Methods section. Aryl
sulfate hydrolysis data by Fendler and Fendler ([)10 were obtained at 100
°C, and aryl phosphorothioate data by Hollfelder and Herschlag (O)13 were
obtained at 37 °C. The phenol leaving groups and their pKa values for the
(9) series are: 3,4-dinitro (5.42), 4-nitro (7.14), 4-chloro, 3-nitro (7.78),
and 4-cyano (7.95); for the ([) series: 2,5-dinitro (5.22), 4-nitro (7.14),
and 3-nitro (8.35); for the (O) series: 3,4-dinitro (5.42), 4-nitro (7.14), and
3-nitro (8.35). The solid lines represent the least-squares fit to the individual
data series with slopes (âlg) of -1.23 ( 0.04 (9), -1.15 ( 0.01 ([), and
-1.15 ( 0.07 (O). All fits gave R2 > 0.99.
Table 1. Rate Constants for Nonenzymatic ArylSulfate Hydrolysis
at 90 °C, pH 10 (see Methods section)
1
aryl substituent
phenol pKa
[substrate] (mM)
k
water (s-
)
3,4-dinitro
4-nitro
4-chloro, 3-nitro
4-cyano
5.42
7.14
7.78
7.95
5-50
1.4 × 10-4
1.0 × 10-6
1.9 × 10-7
1.0 × 10-7
1-10
12.5-100
2.5-25
Table 2. Rate Constants for AP-Catalyzed Aryl Sulfate Hydrolysis
at 60 °C, pH 10 (see Methods section)
1
cat/KM (M-1 s-
)
Figure 3. Rate constant for AP-catalyzed hydrolysis of aryl sulfates as a
function of the rate constant for aryl phosphorothioates with the same leaving
group. Data taken from Figure 2. The solid line represents the best least-
squares fit to the data, with a slope of 1.00 ( 0.08. The open symbols
represent the points for 4-nitrophenyl and 4-cyanophenyl substituents and
are included in the fit.13
aryl substituent
phenol pKa
[substrate] (mM)
k
4-nitro
4-chloro, 3-nitro
4-cyano
7.14
7.78
7.95
8.35
8.55
9.02
9.11
9.29
9.37
9.38
9.95
9.95
10.21
0.5-40
2.5-20
5-40
1.4 × 10-2
6.3 × 10-2
7.6 × 10-3
2.4 × 10-2
5.4 × 10-3
1.1 × 10-2
3.2 × 10-2
4.1 × 10-3
4.9 × 10-3
3.8 × 10-3
1.1 × 10-3
2.0 × 10-3
4.9 × 10-4
3-nitro
10-55
10-110
8-90
3,4-dichloro
3-chloro
3-bromo
3-fluoro
4-bromo
4-chloro
4-fluoro
suggests that the transition states for these enzymatic reactions
are similar.16
8-45
10-110
10-110
8-90
The apparent likeness of the transition states for phosphate
and sulfate AP-catalyzed hydrolysis is most simply accounted
for by model 3 above (Scheme 3C), according to which the
electrostatic interactions in the AP active site have a negligible
effect on charge distribution of the axial bonds in the transition
state for AP-catalyzed reactions. Furthermore, our data cannot
be accounted for by either model 1 or model 2 (Scheme 3A,B).
However, we cannot rule out a coincidental scenario in which
models 1 and 2 hold simultaneously, with forces exerted on
both phosphate and sulfate esters to tighten their transition states
to precisely the same extent. The observed values of âlg are
consistent with those expected based on the solution hydrolysis
values of -1.2 after correction for the stronger nucleophile
10-110
12.5-90
10-110
parent
4-methoxy
obtained for aryl phosphorothioates (Table 2 and Figure 2).13
As noted in the Introduction, AP is much more efficient at
catalyzing the cognate phosphoryl transfer reaction. Neverthe-
less, similar values of âlg of -0.76 ( 0.14 and -0.77 ( 0.10
were obtained for the sulfate and phosphorothioate esters,
respectively (Figure 2). Much of the scatter present in the
individual LFERs in Figure 2 is eliminated by plotting the
logarithm of the rate constant for each aryl sulfate as a function
of the logarithm of the rate constant for the aryl phosphorothio-
ate with the same leaving group (Figure 3). A single line with
a slope of 1.00 ( 0.08 gives a good fit to the data, suggesting
that idiosyncratic binding effects from the aryl substituents are
the main source of scatter and deviations in the enzymatic LFER
plots of Figure 2, as was previously proposed.13,15 This strong
correlation indicates that the âlg values for AP-catalyzed
phosphate and sulfate hydrolysis are virtually identical and thus
(15) (a) O’Brien, P. J.; Herschlag, D. J. Am. Chem. Soc. 1999, 121, 11022. (b)
O’Brien, P. J.; Herschlag, D. Biochemistry 2002, 41, 3207.
(16) The looseness or tightness of a transition state is determined by the bonding
to both the departing and incoming groups. Herein we have measured âlg
and not ânuc, so that we have an experimental measure of bond cleavage
but not bond formation. However, the AP-catalyzed reactions are highly
symmetrical (Scheme 2), having an oxyanionic nucleophile and leaving
group of similar pKa’s; LFERs for nonenzymatic reactions suggest that
differences in the pKa values of this order for the incoming and leaving
groups do not lead to large transition-state asymmetries (refs 17 and 18).
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J. AM. CHEM. SOC. VOL. 126, NO. 38, 2004 11817

A R T I C L E S
Scheme 4
Nikolic-Hughes et al.
present and binding interactions of both bridging and nonbridg-
ing oxygens with the active site Zn2+ ions, as described
previously.15b On the basis of the above observations and
considerations, we conclude that model 3 is most likely.
There are several possible explanations for the absence of
an effect of electrostatic interactions on transition-state nature.
These are described below.
covalent electronic structure of the transition state. This
viewpoint is supported by previous observations in model
systems. Mg2+ or Ca2+ bound to phosphoanhydrides such as
ATP, phosphate monoesters, or phosphorylated pyridines in
aqueous reactions does not tighten the transition state.17
Similarly, the transition states for hydrolysis of phosphates by
E. coli alkaline phosphatase (AP) and Yersinia protein tyrosine
phosphatase are unchanged upon removal of positively charged
active site arginine residues, as judged by LFERs and isotope
effects, respectively.14,15a,21 These observations suggest that
electrostatic interactions with positively charged groups do not
have large effects on the nature of transition state for phosphoryl
transfer, consistent with model 3 and the results herein (Scheme
3C). This conclusion is also consistent with previous substituent
effect studies in model systems that implied that the transition
state is rather difficult to change because the energy surface
near the transition state is steep.17
The classical view of reactions proceeding via loose, meta-
phosphate-like transition states is that charge donation from the
nonbridging oxygen atoms helps expel the leaving group
(Scheme 4A). From this standpoint, electrostatic interactions
with the nonbridging oxygen atoms would inhibit this charge
donation. However, the charge distribution is not known, and
computational work has suggested that metaphosphate may be
better described by a resonance form with one or two positive
charges on the phosphorus atom so that charge donation from
the nonbridging phosphoryl oxygen atoms may not occur in
the transition state (Scheme 4B,C).3b,19 It is also possible that
the negative charge is not evenly distributed among the
nonbridging phosphoryl oxygen atoms, but rather rearranges to
accommodate differential positive potentials within active sites.20
Although addition of covalent bonds to the phosphoryl oxygen
atoms to give phosphate di- and triesters tightens the transition
state, ionic interactions with metal ions or side chains are
expected to be weaker and give less of a perturbation to the
It is also possible that other electrostatic interactions limit
charge flow to the nonbridging oxygen atoms (Scheme 5; dots
depict the strength of electrotatic interactions). As noted above,
the interaction of the nonbridging oxygen atoms with the Zn2+
ions and positively charged Arg side chain (Scheme 2) presum-
ably provides a driving force for accumulation of additional
charge on these oxygen atoms. However, as the incoming and
outgoing oxygen atoms also interact with the active site Zn2+
ions (Scheme 2),6 loss of charge on these atoms would weaken
their electrostatic interactions. Thus, a balance of interactions,
along with any barrier for electron rearrangement from the
otherwise most stable transition state (see Introduction), may
render formation of the unperturbed transition state most
(17) (a) Herschlag, D.; Jencks, W. P. J. Am. Chem. Soc. 1987, 109, 4665. (b)
Herschlag, D.; Jencks, W. P. Biochemistry 1990, 29, 5172. (c) Admiraal,
S. J.; Herschlag, D. Chem. Biol. 1995, 2, 729.
(18) Skoog, M. T.; Jencks, W. P. J. Am. Chem. Soc. 1984, 106, 7597.
(19) (a) Rajca, A.; Rice, J.; Streitweiser, A.; Schaefer, H. J. Am. Chem. Soc.
1987, 109, 4189. (b) Horn, H.; Alhrichs, R. J. Am. Chem. Soc. 1990, 112,
2121.
(20) (a) Spiro, T. G. In Inorganic Biochemistry; Eichhorn, G. L., Ed.; Elsevier:
Amsterdam, 1973; Vol. 1, pp 549-581. (b) Baraniak, J.; Frey, P. A. J.
Am. Chem. Soc. 1988, 110, 4059.
(21) Hoff, R. H.; Wu, L.; Zhou, B.; Zhang, Z.; Hengge, A. C. J. Am. Chem.
Soc. 1999, 121, 9514.
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Interactions with Positive Active Site Groups
A R T I C L E S
Scheme 5. Possible Balance of Electrostatic Interactions in the AP Active Site
Enzymatic Sulfate Ester Hydrolysis Reactions. Standard re-
favorable in the AP active site. Also, the absence of a change
action conditions were 0.1 M NaCHES, pH 10.0, 0.5 M NaCl, 10 mM
MgCl2, and 1 mM ZnSO4 at 60 °C. The appearance of the phenolate
product was monitored noncontinuously using a Uvikon XL spectro-
photometer at the following wavelengths: 4-nitro (410 nm), 4-chloro,
3-nitro (403 nm), 4-cyano (300 nm), 3-nitro (400 nm), 3,4-dichloro
(314 nm), 3-chloro (292 nm), 3-bromo (293 nm), 3-fluoro (283 nm),
4-bromo (292 nm), 4-chloro (298 nm), 4-fluoro (293 nm), parent (292
nm), and 4-methoxy (300 nm). For hydrolysis of 3,4-dichloro-,
3-chloro-, 3-fluoro-, 4-bromo-, and 4-chloro-phenyl sulfate, aliquots
were taken from reactions and diluted 10-fold for absorbance readings,
whereas for all the other reactions absorbance measurements were
obtained without dilution. Rate constants were obtained from initial
rates (e5% reaction). Product formation was linear in all cases, and
AP was shown to retain >80% of the activity during the course of the
slowest reactions (∼1 week). Reactions were shown to be first-order
in substrate and enzyme by varying substrate concentration over a range
of 6-80-fold (Table 2) and enzyme concentration from 1 to 5 µM.
The apparent second-order rate constants, kcat/KM, obtained are reported
per active site in Table 2.
in transition-state structure upon removal of the active site Arg
residue, as determined by âlg, provides no indication of an
electronic rearrangement of the transition state induced by
Arg;14,15a as Arg only interacts with the nonbridging oxygen
atoms, its effect would not be subject to the balance of
interactions.
In conclusion, it appears that the interactions in the AP active
site stabilize the transition state found in the solution reaction,
so that AP uses the minimal amount of energy to achieve
considerable rate enhancements.5,8,15b The challenge we now
face is to understand how these active site interactions cause
the observed stabilization of more than 25 kcal/mol for the
phosphoryl transfer reaction, and what active site features are
responsible for the enormous discrimination exhibited against
the sulfate ester transition state.5,8,15b
Methods
Nonenzymatic Sulfate Ester Hydrolysis Reactions. Standard
reaction conditions were 0.1 M NaCHES, pH 10.0, 0.5 M NaCl, and
90 °C. The reaction was followed in the pH-independent region, to
ensure that S-O bond cleavage was monitored (see ref 9c). The
appearance of the phenolate product was monitored noncontinuously
using a Uvikon XL spectrophotometer at the following wavelengths:
3,4-dinitro (403 nm), 4-nitro (410 nm), 4-chloro, 3-nitro (403 nm), and
4-cyano (300 nm). Rate constants, kwater, were determined from initial
rates (e2% reaction), and the reactions were shown to be first-order
in substrate by varying concentration 8-10-fold (Table 1).
Acknowledgment. We are grateful to P. Dervan for gener-
ously sharing laboratory space and equipment for the aryl sulfate
syntheses, A. Heckel and P. Arora for synthetic advice, J.
Zalatan for helpful discussions, P. O’Brien and members of the
Herschlag and Rees labs for comments on the manuscript. This
work was funded by grants from the NIH to D.H. (GM64798)
and D.C.R. (GM45162). I.N-H. is an AHA Predoctoral Fellow.
JA0480421
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J. AM. CHEM. SOC. VOL. 126, NO. 38, 2004 11819