Catalytic proficiency: The extreme case of S-O cleaving sulfatases

Article abstract of DOI:10.1021/ja208827q

As benchmarks for judging the catalytic power of sulfate monoesterases, we sought to determine the rates of spontaneous hydrolysis of unactivated alkyl sulfate monoesters by S-O bond cleavage. Neopentyl sulfate proved to be unsuitable for this purpose, since it was found to undergo hydrolysis by a C-O bond cleaving mechanism with rearrangement of its carbon skeleton. Instead, we examined the temperature dependence of the spontaneous hydrolyses of aryl sulfate monoesters, which proceed by S-O cleavage. Extrapolation of a Bronsted plot [log(k25^N) = (-1.81 ± 0.09) pK_a^LG + (3.6 ± 0.7)] based on the rate constants at 25 °C for hydrolysis of a series of sulfate monoesters to a pK_a^LG value of 16.1, typical of an aliphatic alcohol, yields k25^N = 3 × 10 ^-26 s^-1. Comparison of that value with established k _cat values of bacterial sulfatases indicates that these enzymes produce rate enhancements (k_cat/k_uncat) of up to 2 × 10²⁶-fold for the hydrolysis of sulfate monoesters. These rate enhancements surpass by several orders of magnitude the ～10 ²¹-fold rate enhancements that are generated by phosphohydrolases, the most powerful biological catalysts previously known. The hydrolytic rates of phosphate and sulfate monoesters are compared directly, and the misleading impression that the two classes of ester are of similar reactivity is dispelled.

Full text of DOI:10.1021/ja208827q

ARTICLE
pubs.acs.org/JACS
Catalytic Proficiency: The Extreme Case of SꢀO Cleaving Sulfatases
David R. Edwards,* Danielle C. Lohman, and Richard Wolfenden*
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, United States
S Supporting Information
b
ABSTRACT: As benchmarks for judging the catalytic power of sulfate monoesterases, we sought
to determine the rates of spontaneous hydrolysis of unactivated alkyl sulfate monoesters by SꢀO
bond cleavage. Neopentyl sulfate proved to be unsuitable for this purpose, since it was found to
undergo hydrolysis by a CꢀO bond cleaving mechanism with rearrangement of its carbon
skeleton. Instead, we examined the temperature dependence of the spontaneous hydrolyses of
aryl sulfate monoesters, which proceed by SꢀO cleavage. Extrapolation of a Bronsted plot
[log(k25N) = (ꢀ1.81 ( 0.09) pKaLG + (3.6 ( 0.7)] based on the rate constants at 25 °C for hydrolysis of a series of sulfate
monoesters to a pKaLG value of 16.1, typical of an aliphatic alcohol, yields k25N = 3 ꢁ 10ꢀ26 sꢀ1. Comparison of that value with
established kcat values of bacterial sulfatases indicates that these enzymes produce rate enhancements (kcat/kuncat) of up to 2 ꢁ 1026-
fold for the hydrolysis of sulfate monoesters. These rate enhancements surpass by several orders of magnitude the ∼1021-fold rate
enhancements that are generated by phosphohydrolases, the most powerful biological catalysts previously known. The hydrolytic
rates of phosphate and sulfate monoesters are compared directly, and the misleading impression that the two classes of ester are of
similar reactivity is dispelled.
’ INTRODUCTION
Sulfuryl transfer is involved in regulating the binding affinity
between receptor and ligand complexes, the control of hormone
levels, detoxification, and other biological activities.1,2 Together,
sulfotransferases and sulfatases control the state of sulfation of
proteins, carbohydrates, lipids, and steroids.1,2 In addition to
their involvement in these natural processes, organososulfate
esters enter the environment as xenobiotics in household and
industrial waste, mainly in the form of surfactants.3 It has been
Figure 1. Chemical structures of sulfate monoesters 1 and 2.
estimated that organosulfates contribute as much as 30% to the
total organic mass found in atmospheric aerosols4 and that they
constitute 50% of the total sulfur content in soil samples.5
An improved understanding of the susceptibility of organo-
sulfate esters to hydrolysis is needed for the design of new
surfactants with desirable properties,6 for the development of
mechanism-based inhibitors of sulfatase/sulfotransferase en-
zymes in disease,7 and to broaden our appreciation of hydrolytic
reaction mechanisms in general.
monoesters by the SꢀO bond cleavage mechanism employed
by sulfatases. Initially, we examined neopentyl sulfate (1a) as a
model substrate, because the steric bulk of the neopentyl
skeleton was expected to disfavor nucleophilic attack at
carbon and because the absence of β-hydrogen atoms pre-
cludes the formal elimination of HSO4ꢀ (Figure 1). However,
1a was found to undergo hydrolysis by a CꢀO cleaving
mechanism with rearrangement of its carbon skeleton. To
circumvent that difficulty, we turned to the hydrolysis at
elevated temperatures of sulfate esters of a series of aryl
alcohols of progressively decreasing acidity, using the Eyring
equation to estimate their reaction rates at ordinary tempera-
tures. A Brønsted analysis of the results leads to the conclusion
that hydrolysis of alkyl sulfate monoesters by SꢀO bond
cleavage is an extraordinarily slow process and that the
enzymes that promote these reactions are the most proficient
biological catalysts identified to date. A kinetic rationale for
the use of a formyl glycine nucleophile by aryl sulfatase
enzymes is proposed.
Sulfate monoesters react with nucleophiles in aqueous solu-
tion to generate products derived from either SꢀO bond cleav-
age8 or CꢀO bond cleavage.9 Most sulfatases catalyze the
hydrolysis of bonds of this type by SꢀO bond cleavage.1 The
spontaneous or pH-neutral hydrolysis of sulfate monoesters
by SꢀO bond cleavage is known to be slow at ordinary
temperatures. Thus, 4-nitrophenyl sulfate (2d), which might
be considered an activated ester based on the acidity of the
product alcohol (pKaLG = 7.14), is hydrolyzed with a half-life
of 45 years at 25 °C at pH values near neutrality.8 Rates of
SꢀO cleavage of less activated sulfate monoesters, with
LG
leaving group pKa values much greater than ∼7, do not
appear to have been reported.10
In the work described here, we set out to determine the rates
of spontaneous hydrolysis of simple unactivated alkyl sulfate
Received: September 19, 2011
Published: November 16, 2011
r
2011 American Chemical Society
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Figure 2. Hydrolysis of neopentyl sulfate (1a) forming a pH dependent
mixture of 3 and 4.
’ EXPERIMENTAL SECTION
Sulfate esters 1a,b and 2c,fꢀj were prepared by standard procedures
as detailed in the Supporting Information. Other reagents were obtained
from commercial sources and used as supplied. Deionized water
(0.11 μS/cm) was used for all kinetic experiments.
Hydrolytic reactions were conducted in quartz tubes sealed under
vacuum. The reaction vessels were placed in thermally equilibrated
ovens equipped with ASTM thermometers for a set time period.
Standard kinetic runs were carried out with 2.0 ꢁ 10ꢀ2 M sulfate
monoester using 0.4 M of the described buffer system to maintain
reaction pH. Typically the 0.2 mL reaction volume was divided into two
portions. One portion was diluted 5-fold with H2O and the pH
measured using an Accumet standard glass combination electrode
(Fisher Scientific). The second portion was diluted 5-fold with D2O
for 1H NMR analysis run on an Inova 500 MHz spectrometer equipped
with a 5 mm cold probe, using a 30 s delay between pulses. Reaction
progress was determined by 1H NMR for a given experiment at a single
time point by comparison of the normalized integrated signal intensities
corresponding to the starting material and reaction products. Observed
first order rate constants were calculated using a standard first order
exponential equation. First order behavior was verified by comparison of
the computed rate constants determined at different time points for
separate reactions run under otherwise identical conditions. Reactions
were free of byproduct and obeyed good first order behavior for >90%
conversion. pKa data for the aryl alcohol leaving groups of 2 were
obtained from literature sources.11 The pKaLG for 1b was estimated to be
16.1 using a σ* of ꢀ0.13 by the method of Ballinger and Long.12 17O
NMR spectra were acquired on a 600 MHz Inova spectrometer
operating at a frequency of 81.31 MHz using a 100 ms repetition rate.
Figure 3. Plot of log(kobs) versus pH for the conversion of 1a into
o
1
a variable mixture of 3 and 4 at 180 C as determined by H NMR
(500 MHz, D2O). The kinetic constants kH = (2.3 ( 0.4) ꢁ 10ꢀ1 Mꢀ1
s
ꢀ1 and kN = (2.7 ( 0.3) ꢁ 10ꢀ6 sꢀ1 were determined from a fit of the
data to eq 1.
of 3 and 4, were determined by 1H NMR (500 MHz, D2O) from
pH 0.7ꢀ10.3 at 180 °C (Figure 3). In these experiments, pH was
generally maintained constant over the course of the reaction
with potassium formate, acetate or phosphate buffers (0.4 M).13
Control experiments using different concentrations of these
buffer systems at constant pH showed small (<15%) effects on
the computed observed rate constants. The hydrolysis of 1a at
pH 0.7 was too fast to measure at 180 °C, and was estimated by
extrapolation of an Eyring plot determined over the range T =
60ꢀ130 °C in the presence of 2.0 ꢁ 10ꢀ1 M HCl (vide infra).
A fit of the kinetic data to eq 1 yielded kH = (2.3 ( 0.4) ꢁ
10ꢀ1 Mꢀ1 sꢀ1 for the acid-catalyzed formation of 3 and kN
=
(2.7 ( 0.3) ꢁ 10ꢀ6 sꢀ1 for the neutral production of 4 (Figures 2
and 3). The ratio of rate constants for the neutral and acid
catalyzed hydrolysis of 1a is such that equal amounts of 3 and 4
are formed at the near neutral pH of 4.9.14
logðk Þ ¼ logðkH ꢁ 10ꢀpH þ kNÞ
ð1Þ
obs
’ RESULTS
C. Activation Parameters for the Hydrolysis of 1a and 1b.
Sulfate ester 1a was hydrolyzed in the presence of potassium
phosphate buffer (0.4 M, pH 8.1) over the temperature range
spanning 160ꢀ243 °C. Fitting the kinetic data to the Eyring
equation yielded ΔH‡ = 23.0 ( 1.3 kcal/mol and ΔS‡ = ꢀ33 (
A. Hydrolysis of Neopentyl Sulfate (1a). A 200 μL aqueous
solution containing 2.0 ꢁ 10ꢀ2 M 1a and 0.4 M formate buffer at
pH 4.05 was sealed in a quartz tube under vacuum and heated to
180 °C for 110 min. Following reaction, the sealed quartz tube
was cooled in an ice bath and the contents diluted 5-fold with
D2O for analysis by 1H NMR using a standard water suppression
2.5 cal/mol K. Extrapolation of the Eyring plot led to an
3
estimated rate constant of 5 ꢁ 10ꢀ12 sꢀ1 at 25 °C, corresponding
to t1/2 > 4000 years for the neutral hydrolysis of 1a (Table 1).
Sulfate ester 1a was also subjected to hydrolysis in 2.0 ꢁ 10ꢀ1 M
HCl (pH 0.7) at temperatures ranging from 60 to 130 °C, and
second order rate constants were calculated as k2H = kobs/[H+].
The activation parameters ΔH‡ = 31.5 ( 0.05 kcal/mol and
ΔS‡ = +7.3 ( 0.1 cal/mol.K were determined by fitting the
kinetic results to the Eyring equation. Extrapolation of the Eyring
plot led to an observed first order rate constant of 1.9 ꢁ 10ꢀ9 sꢀ1
(t1/2 = ∼12 years) in 1 M HCl and 25 °C. For comparison,
n-pentyl sulfate (1b) was hydrolyzed from 94 to 185 °C in the
presence of a 0.2 M ethyl phosphonic acid buffer at pH 7.4, where
the hydrolysis of 1b was independent of pH and buffer catalysis
was found to be negligible. A fit of the rate data to the Eyring
equation yields ΔH‡ = 25.2 ( 1.9 kcal/mol and ΔS‡ = ꢀ25 (
4 cal/mol.K for the pH neutral hydrolysis of 1b (Table 1).
1
sequence. H NMR (500 MHz, D2O) of the crude reaction
mixture revealed the presence of unreacted 1a and neopentanol
(3) in a ratio of 82:18 as determined by comparison to authentic
samples (Figure 2). An observed rate constant of 3.0 ꢁ 10ꢀ5 sꢀ1
was calculated from the integrated signal intensities using a
standard first order exponential equation. In a similar manner,
1a was hydrolyzed at pH 7.6 maintained with potassium phos-
phate buffer (0.4 M) at 180 °C for 17.8 h. The 1H NMR spec-
trum of this product mixture contained three new sets of peaks,
corresponding to tert-amyl alcohol (4), in addition to unreacted
1a (molar ratio of 1.1: 4.7). The observed rate constant for
conversion of 1a to 4 under these conditions was 3.3 ꢁ 10ꢀ6 sꢀ1
(Figure 2).
B. pH Dependence of the Hydrolysis of 1a at 180 °C. Rate
constants for the disappearance of 1a, forming a variable mixture
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Table 1. Activation Parameters and Computed First Order
Rate Constants, kN25, at 25 oC for the Neutral Hydrolysis of 1a,
b and 2aꢀj
ΔH‡
pKLaG (kcal/mol) (cal/mol/K)
CꢀO bond cleavage
ΔS‡
a
1/2b
kN25 (sꢀ1
)
t
25
1a
1b 16.1
15.5c
23.0 ( 1.3
25.2 ( 1.9
ꢀ33 ( 2.5
ꢀ25 ( 4
5.0 ꢁ 10ꢀ12 4.4 ꢁ 103 yrs
6.8 ꢁ 10ꢀ12 3.2 ꢁ 103 yrs
SꢀO bond cleavage
2ad
2bd
2c
4.11
18.2
ꢀ18.0
(4.0 ꢁ 10ꢀ5 d
)
6 h
5.04
5.49
7.14
7.21
8.38
9.02
9.38
9.99
18.8
ꢀ17.4
1.5 ꢁ 10ꢀ5
13 h
ND
ND
(9.9 ꢁ 10ꢀ8 e
)
85 days
2d
2ed
2f
25.9 ( 1.2
24.1
ꢀ16 ( 3
ꢀ17.4
1.9 ꢁ 10ꢀ10
2.0 ꢁ 10ꢀ9
6.7 ꢁ 10ꢀ12
6.3 ꢁ 10ꢀ13
1.3 ꢁ 10ꢀ14
2.3 ꢁ 10ꢀ15
1.1 ꢁ 10ꢀ16
45 yrs
11 yrs
26.7 ( 1.3
27.2 ( 0.9
32.3 ( 1.9
32.9 ( 1.0
35 ( 5
ꢀ20 ( 4
ꢀ23 ( 2
ꢀ14 ( 4
ꢀ15 ( 2
ꢀ14 ( 9
1.2 ꢁ 103 yrs
3.5 ꢁ 104 yrs
1.7 ꢁ 106 yrs
1.6 ꢁ 107 yrs
2.0 ꢁ 108 yrs
2g
2h
2i
Figure 4. 17O NMR (81.4 MHz, D2O) spectra of (a) recovered sulfate
following the acid catalyzed hydrolysis of 1a at 150 oC where the peak at
167 ppm corresponds to 17O-sulfate and; (b) recovered sulfate following
hydrolysis of 1a at pH 8.4 (0.1 M phosphate buffer), 200 oC where the
peakat94 ppm corresponds to17O-phosphate. The spectraare referenced
to D2O at 0 ppm. The spectra in (a) and (b) were acquired with 256 and
1024 scans, respectively.
2j
10.6
a kN25 computed from the activation parameters listed in the table using
the Eyring equation unless otherwise noted. b Calculated from k2N5
and a standard first order exponential equation. c The pKaLG for 1a has
previously been assumed to be approximately 15.5 (ref 22), and we
find this value to be sufficiently accurate for our limited purposes of
providing a visual reactivity comparison in Figures 7 and Supporting
Information Figure S11. d Data from ref 10. e k2N5 for 2c represents an
observed first order rate constant determined by initial rates at pH 8.5
and 25 oC. Activation parameters were not determined for hydrolysis
of this substrate. A standard error of (5% was determined from
duplicate runs.
Figure 5. Proposed reaction mechanism for the acid catalyzed hydro-
lysis of 1a.
D. 17O Labeling Experiments for Hydrolysis of 1a and 1b.
An isotope labeling experiment was conducted to identify the
position of bond cleavage during the hydrolysis of 1a. A solution
(0.05 M) of 1a at an initial pH of 2.85 (1.4 ꢁ 10ꢀ3 M HCl) was
hydrolyzed at 150 °C in water containing 6.1 atom % excess 17O
for 20 h, at which time the hydrolysis of 1a was >99% complete.
After concentration to dryness at reduced pressure and recon-
stitution in D2O to remove residual H217O,15 analysis by 17O
NMR (81.4 MHz, D2O) was carried out on a sample containing
triethylammonium sulfate (0.015 M).16 The peak at 167 ppm
shown in Figure 4a corresponds to 17O-sulfate, and by compar-
ison to the intensity of the solvent peak we estimate 6.7%
incorporation of the label. We interpret this result as consistent
pH 8.4 (Supporting Information). Under these conditions, the
hydrolytic reactions were shown to be independent of hydro-
nium ion concentration and free of observable buffer effects.
Activation parameters for the pH neutral hydrolysis of 2d,fꢀj
were determined by fitting the rate data to the Eyring equation
and are shown in Table 1 along with results for 2a, 2b, and 2e
obtained from various sources.8,10 The activation parameters
previously reported for the hydrolysis of 2d are ΔH‡ = 24.4 kcal/
mol (ΔS‡ = ꢀ19.5 cal/mol/K) and ΔH‡ = 24.6 kcal/mol
(ΔS‡ = ꢀ18.5 cal/mol/K) in satisfactory agreement with those
determined here.8,17 Rate data for 2c were obtained at 25 °C and
pH 8.4 by the method of initial rates, using UV spectrophotom-
etery to monitor the release of pentafluorophenoxide at 260 nm
(effective ε260 = 1023).
2‑
with quantitative incorporation of the 17O-label into SO4 as
would be expected for an SꢀO bond cleaving mechanism. In a
similar manner, 1a was hydrolyzed at 200 °C and pH 8.4 (0.1 M
phosphate) for >5 half-lives in water containing 6.1% H217O.
The 17O NMR of the product mixture of this reaction showed a
broad peak at 94 ppm determined to be the inorganic phosphate
used as buffer in the reaction, but no peak corresponding to
inorganic sulfate could be detected (Figure 4b). We estimate
the lower limit of detection using this method as 1 ꢁ 10ꢀ4 M,
indicating that at least 90% of the pH neutral hydrolysis of 1a
occurs by CꢀO bond fission. Hydrolysis of 1b at pH 8.4 and
200 °C for 20 h led to identical results, consistent with a CꢀO
bond cleavage of this substrate as well.
’ DISCUSSION
A. Acid Catalyzed Hydrolysis of 1a. A mechanism consistent
with the available data for the acid catalyzed hydrolysis of 1a is
shown in Figure 5 involving a pre-equilibrium proton transfer
from solvent to a nonbridging oxygen atom of the sulfate
monoester. The pKa of 1a can be estimated as ꢀ3.4,18 so the
equilibrium shown in Figure 7 is expected to lie heavily toward
the anionic sulfate monoester under the pH conditions of the
current study. Heavy atom kinetic isotope effects on the acid
catalyzed hydrolysis of 4-nitrophenyl sulfate support the transfer
E. Hydrolysis of Aryl Sulfate Monoesters. Aryl sulfate
monoesters 2d,fꢀj were hydrolyzed at elevated temperatures,
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Figure 6. Proposed reaction mechanism for pH neutral hydrolysis of 1a
to form tert-amyl alcohol.
of the SꢀOH proton to the leaving group through a network of
one or more water molecules concerted with SꢀO bond
cleavage.17 It seems reasonable to suppose that a similar network
of solvent molecules acts on alkyl sulfate 1a, but there is evidence
that the steric bulk of the neopentyl substituent may also disrupt
the solvent shell surrounding the activated complex for reaction
of this substrate. First, a Brønsted plot of the second order rate
constants for the acid catalyzed hydrolysis of 5 aryl and 2 primary
alkyl sulfate monoesters at 25 °C (Figure S11, Supporting
Information)9,10,19 fits the equation:
Figure 7. Brønsted plot of log(kN25) versus pKaLG for the hydrolysis of
sulfate monoesters 2aꢀj (9) at 25 oC fit to a standard linear regression
of log(kN25) = (ꢀ1.81 ( 0.09) pKaLG + (3.6 ( 0.7), r2 = 0.9801 (10 data).
The experimental data for the neutral hydrolysis of 1a (red cross) and 1b
(red solid circle) are shown for comparison along with the predicted rate
constants for hydrolysis proceeding through SꢀO bond cleavage (open
red square). The latter two data points are computed from the linear
regression and have not been included in the fit.
logðkH2 Þ ¼ ðꢀ 0:33 ( 0:01ÞpKa ꢀ ð2:5 ( 0:1Þ
LG
Significantly, the point corresponding to 1a lies approximately
one logarithmic unit below the best fit line. Second, the entropy
of activation for the acid catalyzed hydrolysis of the same five
aryl and two alkyl sulfate monoesters is effectively substrate-
independent, with an average ΔS‡ of ꢀ3.4 ( 3 cal/mol/K,
whereas ΔS‡ for the acid catalyzed hydrolysis 1a is distinctly
more positive (+7.3 ( 0.2 cal/mol/K). The acid catalyzed hydro-
lysis of 1a is slower than expected relative to a suitable compar-
ison provided by the Brønsted correlation and also involves a
small but significantly increased activation entropy compared
with the reactions of the other sulfate monoesters. These two
observations would seem understandable if the large neopentyl
substituent disrupted interactions with the surrounding solvent
shell that would otherwise stabilize the activated complex through
proton transfer relays and other polar interactions.
Earlier experiments in this laboratory9 showed that a broad
range of nucleophiles (1.72 e pKaNu e 15.7) are alkylated by
methyl sulfate in aqueous solution, furnishing a precedent for
attack by weak nucleophiles, such as water, on alkyl sulfate
monoesters. In fact methyl sulfate was found to be some 100-
fold more reactive in this regard than the trimethylsulfonium ion.
Consistent with these findings, 1b undergoes spontaneous
hydrolysis in the pH range 6ꢀ8 by attack of water on carbon
2‑
to displace SO4
.
C. Spontaneous Hydrolysis of Aryl Sulfate Monoesters.
Rate constants, kN25, for the reaction of 2 at 25 °C can be
calculated using the activation parameters in Table 1.24 Notably,
these are seen to vary by a factor of more than 1011 from the most
reactive substrate (2a) to the least reactive (2j). The variation in
reactivity of 2aꢀj arises from the steep dependence of ΔH‡ on
the ionization constants, pKaLG, of the conjugate acids of the
aryloxy leaving groups.25 The ΔS‡ values all fall within the
relatively narrow range of ꢀ17 ( 3 cal/mol/K.
B. Spontaneous Hydrolysis of 1a/1b. The pH-independent
hydrolysis of 1a does not conform to the typical reaction mecha-
nisms observed for sulfate monoesters, as evidenced by the
rearrangement of the neopentyl carbon skeleton to form tert-
amyl alcohol (4). The results of the 17O labeling study support a
CꢀO bond cleaving mechanism such as the one presented in
Figure 6, involving rate-limiting methyl migration with expulsion
of SO42‑. Subsequent capture of cationic intermediate 5 by water
or possibly hydroxide ion would then afford 4. The sterically
demanding neopentyl system has been known for some time to
undergo solvolysis with rearrangement similar to that observed
here, but the leaving groups have typically been highly reactive
sulfonates.20 We were therefore surprised to find that 1a,
A Brønsted plot of log(kN25) versus pKaLG for hydrolysis of
2aꢀj fits a standard linear regression of log(kN25) = (ꢀ1.81 (
0.09) pKaLG + (3.6 ( 0.7) with a Brønsted coefficient of
β
LG = ꢀ1.8 (Figure 7).26 The βLGdetermined here for hydrolysis
of aryl sulfate monoesters at 25 °C is significantly more negative
than that reported for the hydrolysis of a limited set of nitro-
phenyl and dinitrophenyl sulfate monoesters at 100 °C, where
equipped with a formally dianionic leaving group with pKaLG
=
β
LG= ꢀ1.2.10 This discrepancy originates in the steep dependence
1.8,19 also underwent this process. Hydrolysis of the monoanio-
nic 3-(4-carboxyphenyl)-2,2-dimethylpropyl phosphate monoe-
ster has been shown to generate the PꢀO bond cleavage product
cleanly, without formation of detectable amounts of isomerized
product.21 In a similar manner, the neutral hydrolyses of the
monoanionic diesters bis-neopentyl phosphate and bis-
(3-(4-carboxyphenyl)-2,2-dimethylpropyl) phosphate yield only
PꢀO bond cleavage products.22 The observation that 1a pre-
ferentially undergoes unimolecular rearrangement with elimina-
tion of SO42‑, rather than hydrolysis by SꢀO bond rupture, is
consistent with the extreme inertness of this functional group to
the latter mode of cleavage.23
of ΔH‡ on pKaLG for the neutral hydrolysis of aryl sulfate mono-
esters. A decrease in reaction temperature results in the rates of
hydrolysis for the less activatedsubstrates beingdisproportionately
reduced relative to the rates of hydrolysis of the more activated
sulfate esters. The result isa gradual increase inthe steepnessof the
observed Brønsted plot with decreasing temperature.
The equilibrium Brønsted coefficient, βeq, determined for the
transfer of the sulfuryl group between oxyanion nucleophiles at
25 C is ꢀ1.74.27 Accordingly, the large negative βLG of ꢀ1.8
o
observed here indicates that the leaving groups in 2aꢀj have
undergone near complete bond cleavage and bear formal charges
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of approximately ꢀ1 at the transition state for hydrolysis. It
might be suggested on this basis that hydrolysis of aryl sulfate
monoesters proceeds via a fully dissociative DN + AN mechanism.
However, the Brønsted coefficient, βLG, reports solely on the
extent of bond cleavage to the leaving group and therefore cannot
be used to distinguish a DN + AN mechanism from an dissociative
ANDN process involving little bond formation to a nucleophilic
water molecule. Based on heavy atom kinetic isotope effects,
Hengge et al. have described the hydrolysis of 4-nitrophenyl
rate enhancement (kcat/knon) of 2 ꢁ 1026-fold, or a catalytic
proficiency (kcat/Km)/kuncat) of 1 ꢁ 1029 Mꢀ1 are calculated.
Another SꢀO bond cleaving sulfatase, the D-lactate-2-sulfatase
(EC 3.1.6.17) from Pseudomonas syringae GG, catalyzes the hydro-
lysis of its preferred substrate with kcat = 13.1 sꢀ1 and kcat/Km =
2.0 ꢁ 103 Mꢀ1 sꢀ1 30
.
Extrapolation of the Brønsted plot in
Figure 7 to a pKaLG of 15.131 indicates a first order rate constant of
2.0 ꢁ 10ꢀ24 sꢀ1 for the spontaneous hydrolysis of lactate-2-sulfate.
A rate enhancement of kcat/kuncat = 7 ꢁ 1024ꢀfold and a catalytic
sulfate as involving an exploded00 transition state structure in
proficiency of (kcat/Km)/kuncat = 1 ꢁ 1027
M
ꢀ1 are calculated. A
00
which the sulfur trioxide moiety is only minimally bonded to the
incoming water nucleophile and the departing aryloxide.17 The
linear free energy results presented here support this view, and, in
fact, there is a remarkable agreement regarding the extent of
charge development on the leaving group in the transition state
arising from the two studies. A charge map for the spontaneous
hydrolysis of aryl sulfate monoesters is shown in Figure 8
displaying the formal charge born by the aryloxy group in the
ground, transition and product states.
third example, the Pseudomonas aeruginosa arylsulfatase generates
a 2.3 ꢁ 1010-fold rate enhancement for the hydrolysis of 2d, with a
catalytic proficiency of 1 ꢁ 1017 Mꢀ1 32
.
Most sulfatases have been reported to catalyze the selective
hydrolysis of sulfate monoesters by SꢀO bond fission,1 but a few
prokaryotic sulfatases have been shown to catalyze CꢀO bond
cleavage of alkyl sulfate monoesters. For example, the P2 primary
alkylsulfohydrolase from Pseudomonas C12B catalyzes the hydro-
lysis of 1b at an optimal pH of 8.3 (30 °C) with kcat/Km
=
320 Mꢀ1 sꢀ1 by what is almost certainly a CꢀO bond cleaving
D. Catalytic Proficiency and Rate Enhancements Provided
by Sulfatase Enzymes. Earlier, a rate enhancement (kcat/kuncat
)
mechanism.33 That value indicates a rate enhancement (kcat/kuncat
)
of at least 1011 was estimated for sulfatases, based on the rate of
spontaneous methyl sulfate hydrolysis, without reference to the
site of bond cleavage.9 We are now in a position to offer a more
concrete estimate of the effectiveness of sulfatases that catalyze
SꢀO bond cleavage (Table 2). An alkylsulfatase from the bac-
terium coryneform B1a catalyzes the hydrolysis of linear alkyl
sulfates by SꢀO bond cleavage.28 The reported kinetic para-
meters for the enzymatic hydrolysis of 1b are kcat = 6.9 sꢀ1 and
kcat/Km = 3.6 ꢁ 103 Mꢀ1 sꢀ1 at pH 7.5. Rate constants for the
spontaneous hydrolysis of 1b by SꢀO bond cleavage are not
directly accessible by experiment. However, a first order rate
constant of 3 ꢁ 10ꢀ26 sꢀ1 can be estimated by extrapolation of
the Brønsted plot in Figure 7 to a pKaLG of 16.1 corresponding to
n-pentanol under the reasonable assumption that the correla-
tion obtains for both alkyl and aryl sulfate esters.29 An enzymatic
of 7 ꢁ 1011 and a catalytic proficiency ((kcat/Km)/kuncat) of
3 ꢁ 1013 Mꢀ1 (Table 2).
E. Comparing the Rates of Hydrolysis for Phosphate and
Sulfate Monoesters. Alkaline phosphatase catalyzes the hydro-
lysis of phosphate monoester dianions via a two-step ping-pong
mechanism involving the formation of a phosphoserine inter-
mediate (eq 2).34 Aryl sulfatases employ a variation on this
mechanism to hydrolyze sulfate monoesters. In this case, the
active site nucleophile is identified to be a formyl glycine (fGly)
residue, which releases inorganic sulfate by CꢀO bond cleavage
in the second step of the reaction (eq 3).35 We rationalize the
different catalytic mechanisms employed by the enzymes by
considering the relative rates of hydrolysis for alkyl phosphate
versus alkyl sulfate monoesters.
Figure 8. Charge map for the pH neutral hydrolysis of aryl sulfate esters
2aꢀj where formal charges on the aryloxy leaving group are indicated in
parentheses.
Table 2. Rate Enhancements and Catalytic Proficiencies of Sulfatases and Phosphatasesa
catalytic proficiency
(kcat/Km)/kuncat (Mꢀ1
rate enhancement
kcat (sꢀ1
)
kcat/Km (Mꢀ1 sꢀ1
)
kuncat (sꢀ1
)
)
kcat/kuncat
sulfatases
P2 primary alkylsulfohydrolaseb
arylsulfatase
9.7
14.2
13.1
6.9
321
1.4 ꢁ 10ꢀ11
4.9 ꢁ 10ꢀ10
2 ꢁ 10ꢀ24
3 ꢁ 10ꢀ26
3 ꢁ 1013
1 ꢁ 1017
1 ꢁ 1027
1 ꢁ 1029
7 ꢁ 1011
3 ꢁ 1010
7 ꢁ 1024
2 ꢁ 1026
4.9 ꢁ 107
2.0 ꢁ 103
3.6 ꢁ 103
D-lactate-2-sulfatase
alkylsulfatase
phosphatases
c
c
c
inositol 1-phosphatase
fructose bisphosphatase
protein phosphatase 1
22
21
39
3 ꢁ 105
1.5 ꢁ 107
4 ꢁ 106
(2 ꢁ 10ꢀ20
(2 ꢁ 10ꢀ20
(2 ꢁ 10ꢀ20
)
)
)
2 ꢁ 1025
8 ꢁ 1026
2 ꢁ 1026
1 ꢁ 1021
1 ꢁ 1021
2 ꢁ 1021
a Data correspond to SꢀO (or PꢀO) bond cleavage unless specified otherwise. b CꢀO bond cleavage. c Data from ref 21.
529
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Journal of the American Chemical Society
ARTICLE
to reaction of the sulfate monoesters is significantly steeper
(βLG= ꢀ1.75) than that for the phosphate monoester dianions
(βLG= ꢀ1.26). The net result, clearly apparent in the Figure, is a
divergence in reactivity for phosphate and sulfate monoesters as
the leaving group becomes less activated (higher pKaLG). The
inset of Figure 9 shows the ∼105 fold difference in reactivity
expected for hydrolysis of alkyl phosphate and sulfate mono-
esters. We propose that this difference in reactivity compels the
aryl sulfatase to seek an alternate mechanism, such as the one
presented in eq 3, to circumvent the difficult task of cleaving an
alkyl sulfo-enzyme intermediate by SꢀO bond cleavage.
’ CONCLUSION
The totem in Figure 10 displays the rate constants (and half-
lives) for the spontaneous hydrolysis of several classes of ester.
The stability of alkyl sulfate monoesters toward hydrolysis exceeds
that of phosphate monoesters by several orders of magnitude. The
enzymes that catalyze hydrolysis of alkyl sulfate monoesters by SꢀO
bond cleavage appear to be the most proficient enzymes identified
to date providing catalytic rate enhancements of up to 1026-fold.
This value surpasses by several orders of magnitude the ∼1021-fold
rate enhancements that are generated by phosphohydrolases, the
most powerful biological catalysts previously known (see Table 2 for
a comparison of catalytic proficiency and rate enhancement pro-
vided by sulfatases and phosphatase).
Figure 9. Brønsted plot of log(kN39) versus pKaLG for the hydrolysis of
sulfate monoesters 2aꢀj (0) and aryl phosphate monoester dianions38
o
(b) at 39 C. Inset: Horizontal axis has been extended so that the
’ ASSOCIATED CONTENT
predicted reactivities of alkyl phosphate and sulfate monoesters can be
compared.
S
Supporting Information. Description of synthetic pro-
b
cedures and analytical data for 1a,b and 2c,fꢀj. Plot of log(kH2 )
versus pKaLG. Plot of log(k2N5) versus pKaLG for the pH neutral
hydrolysis of 2a,c,d. Eyring plots for the hydrolysis of 1a,b and
2d,fꢀj. This material is available free of charge via the Internet at
http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
richard_wolfenden@med.unc.edu; dredward@email.unc.edu
’ ACKNOWLEDGMENT
We thank Greg Young, Facility Manager for the UNC Bio-
molecular NMR laboratory Core Facility, for providing the 17O
NMR spectra. This work was supported by National Institutes of
Health Grant GM-18325.
’ REFERENCES
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o
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530
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Journal of the American Chemical Society
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ꢀ1 was estimated for
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a phosphate and sulfate monoester using n-pentyloxide as the leaving
group with pKaLG of 16.1.
(38) Rate data the hydrolysis of aryl phosphate monoester dianions
taken from refs 22 and 36.
where pH = ꢀlog(Kw
/[ꢀOH]). Thermodynamic data obtained-
453 K
from: Eisenberg, D.; Kauzmann, W. The Structure and Properties of
Water; Oxford University Press: London, 1969.
(14) Neutral pH at 453 K is computed to be ∼5.3 where Kw
=
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[H+][ꢀOH] = (10ꢀ5.3) ꢁ (10ꢀ5.3).
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(24) Rate data for hydrolysis of 2a and 2c at 25 C were available
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ꢀ
(29) The equilibrium ROSO3
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ꢀOH a ROꢀ + HOSO3 is
described by the equation log(K) = ꢀ1.74pKaROH + 28.71 at 25 oC and
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sulfate monoesters.27 It seems reasonable that the kinetic relationship of
log(kN25) = (ꢀ1.81 ( 0.09) pKaLG + (3.6 ( 0.7) determined here will
also apply to both alkyl and aryl sulfates considering the similar formal
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531
dx.doi.org/10.1021/ja208827q |J. Am. Chem. Soc. 2012, 134, 525–531