Mechanistic studies of the hydrolysis of p-nitrophenyl sulfate catalyzed by arylsulfatase from Helix pomatia

Article abstract of DOI:10.1080/00958972.2010.500377

The kinetics of the hydrolysis of small aryl substrate p-nitrophenyl sulfate (p-NPS) catalyzed by arylsulfatase from Helix pomatia was studied at a wide range of temperatures as well as at ambient and elevated pressures. The employed kinetic assay techniq

Full text of DOI:10.1080/00958972.2010.500377

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Mechanistic studies of the hydrolysis
of p-nitrophenyl sulfate catalyzed by
arylsulfatase from Helix pomatia
a
a
Iwona Stawoska , Sylwia Gawęda , Magdalena Bielak-Lakomska
a
a
a
b
,
Małgorzata Brindell , Krzysztof Lewiński , Piotr Laidler &
a
Grażyna Stochel
a
Department of Inorganic Chemistry, Faculty of Chemistry ,
Jagiellonian University , Ingardena 3, 30-060 Kraków, Poland
b
Chair of Medical Biochemistry , Jagiellonian University, Medical
College , Kopernika 7, 31-034 Kraków, Poland
Published online: 13 Jul 2010.
To cite this article: Iwona Stawoska , Sylwia Gawęda , Magdalena Bielak-Lakomska , Małgorzata
Brindell , Krzysztof Lewiński , Piotr Laidler & Grażyna Stochel (2010) Mechanistic studies of the
hydrolysis of p-nitrophenyl sulfate catalyzed by arylsulfatase from Helix pomatia , Journal of
Coordination Chemistry, 63:14-16, 2472-2487, DOI: 10.1080/00958972.2010.500377
To link to this article: http://dx.doi.org/10.1080/00958972.2010.500377
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Journal of Coordination Chemistry
Vol. 63, Nos. 14–16, 20 July–20 August 2010, 2472–2487
Mechanistic studies of the hydrolysis of p-nitrophenyl sulfate
catalyzed by arylsulfatase from Helix pomatia§
IWONA STAWOSKAy, SYLWIA GAWEDAy,
˜
MAGDALENA BIELAK-LAKOMSKAy,
´
MALGORZATA BRINDELLy, KRZYSZTOF LEWINSKIy,
_
PIOTR LAIDLERzand GRAZYNA STOCHEL*y
yDepartment of Inorganic Chemistry, Faculty of Chemistry, Jagiellonian University,
Ingardena 3, 30-060 Krakow, Poland
´
zChair of Medical Biochemistry, Jagiellonian University, Medical College,
Kopernika 7, 31-034 Krakow, Poland
´
(
Received 17 February 2010; in final form 5 May 2010)
The kinetics of the hydrolysis of small aryl substrate p-nitrophenyl sulfate ( p-NPS) catalyzed
by arylsulfatase from Helix pomatia was studied at a wide range of temperatures as well as
at ambient and elevated pressures. The employed kinetic assay techniques allow for the
6
6¼
determination of activation entropy (DS ), activation enthalpy (DH ), as well as activation
6¼
volume (DV ) which suggested associative interchange character of the sulfate ester hydrolysis.
The pH dependence obtained for catalytic parameters suggests that two protonation/
deprotonation reactions can occur and the optimum pH for the catalytic activity was also
detected.
Keywords: Enzymatic hydrolysis; Arylsulfatase from Helix Pomatia; Activation volume;
p-Nitrophenyl sulfate; Desulfatation mechanisms
1. Introduction
Sulfatases (EC 3.1.5.6) have been found in prokaryotic and eukaryotic organisms since
the beginning of the twentieth century, but formerly their role was connected only to the
degradation of organic sulfates in soils [1–3]. Interest in sulfatases rose significantly
when it was discovered that sulfatase deficiencies in human organisms led to various
types of lysosomal disorders. At least eight serious human diseases associated with
the malfunction of single sulfatases have been described [4–6]. In addition, deficiency
of all known sulfatases is a major reason of multiple sulfatase deficiency (MSD), a rare
autosomal recessive disorder [7].
Sulfatases are a conserved family of enzymes, with sequence homology ranging from
0% to 60% and highly conserved N-terminal region carrying motif that is unique
2
for this class of enzymes [8–10]. Arylsulfatases (ARSs) catalyze hydrolytic desulfatation
*
§
Corresponding author. Email: stochel@chemia.uj.edu.pl
On the occasion of Professor Rudi van Eldik’s retirement.
Journal of Coordination Chemistry
ISSN 0095-8972 print/ISSN 1029-0389 online ß 2010 Taylor & Francis
DOI: 10.1080/00958972.2010.500377

Mechanistic studies of the hydrolysis of p-nitrophenyl sulfate
2473
of sulfate esters (CO–S) as well as sulfamates (CN–S) of various substrates, reactions
(
1) and (2), respectively.
ꢀ
ꢀ
4
ROSO þH O !ROH þHSO
ð1Þ
ð2Þ
3
2
ꢀ
ꢀ
4
RNðHÞSO þH2O !RNH2 þHSO
3
ARS from Helix pomatia (Roman snail) [9, 11] comprises 503 amino acids and shows
2% identity to human arylsulfatase B (HARSB) and 27% to human lysosomal
5
arylsulfatase A (HARSA). The residues that are characteristic for the active site
in eukaryotic ARSs are also present in the sequence of sulfatases from H. pomatia.
Results obtained from crystallographic as well as mutagenesis studies of ARSs allowed
detailed characterization of the structure of the active site, which is built by ten polar
aminoacids and a divalent metal ion (table S1 and scheme S1) [9].
Mechanism of sulfatases activity became a subject of intense investigations
when post-translational modification of cysteine (or serine in some prokaryotes) to
formylglycine residue (FGly, 2-amino-3-oxypropanic acid, ꢀ-formylglycine) was
proposed to play an important role in the hydrolysis reaction [10]. This modification
has been found in all known sulfatases and it is now established well that this residue is
crucial for the catalytic activity of the enzyme [12]. The crystal structure of HARSB
determined by Bond et al. [13] gives an insight into the detailed structure of the active
site as well as revealing its structural similarity to alkaline phosphatase (AP). This study
allowed authors to propose an addition-hydrolysis mechanism of sulfate ester
hydrolysis catalyzed by HARSB by referring to the mechanism suggested for phosphate
ester hydrolysis catalyzed by AP [13]. The X-ray structure of HARSA, determined a
year later by Lukatela et al. [14], showed that FGly can serve its catalytic functions only
as a hydrated aldehyde. On the basis of this observation, the authors proposed a novel
transesterification–elimination (TE) mechanism for this reaction [14, 15]. Many studies
based on the crystal structure of HARS and proteinase-activated receptors (PARs), as
well as mutagenesis of HARS, have been conducted in order to elucidate the single steps
underlying the proposed mechanism (an excellent review on this subject is presented
in reference [9], the original work includes references [14–19]). At present, it is believed
that hydrolysis of sulfate ester occurs by TE mechanism as schematically shown in
scheme 1. One of the hydrated groups of formylglycine begins a nucleophilic attack
on the sulfur of the substrate leading to the release of conjugated alcohol and formation
of a covalent FGly-sulfate intermediate (FGS). The final step comprises elimination of
2
4
ꢀ
SO and regeneration of FGly.
According to this mechanism, the configuration of the sulfate is inverted during
hydrolysis since nucleophilic attack and elimination at the sulfate group occur from the
same side [16]. The crystal structure of sulfatase from Pseudomanas aeruginosa (PARs)
revealed that orientation of sulfate is optimal for nucleophilic attack of the FGly
oxygen – Oꢁ1 coordinated to the metal center and Asp317. At the same time, the
oxygen – Oꢁ2 coordinated to His115, Arg55, and Lys113 is crucial for the elimination
of FGS to regenerate FGly [17]. In the studies where FGly was substituted by serine
residue, the modified enzymes were not able to eliminate the sulfur group from FGS,
confirming the importance of non-estrificated hydroxyl of the aldehyde hydrate in rapid
desulfatation of substrate [18]. Evidence confirming the role of gem-diol came from
the crystal structure of HARSA containing formylglycine phosphate derivative.

2
474
I. Stawoska et al.
(a)
(b)
R
+
+
4
O
LysB-NH
H–N-HisB
3
_
O
S
M++
1
+
+
O3
LysB-NH
H–N-HisB
3
O 2
M++
γ2
HO
γ1
OH
γ1
HisA-N
HisA-N
HO
OH
–
–
O-AspC
O-AspC
–
ROSO3
FGly
FGly
–ROH
H O
2
+
N-HisB
LysB-NH3
N-HisB
_
O3
+
M++
1
O2
O
LysB-NH
3
S
H-O-AspC
++
M
+
γ2
HisA-N-H
O
H
HisA-N
HO
O
H–O-AspC
2
–
FGly
–SO
4
FGly
(c)
(
d)
Scheme 1. A TE mechanism of catalytic activity of ARSs proposed on the basis of several independent
studies [9, 14, 15].
The derivative, being a product of inhibition of ARS by phosphate esters, not only
corresponds to one of the intermediates in the postulated mechanism but also shows
that the enzyme has weak phosphate activity [19].
Whereas sulfatases have been a subject of many kinetic studies [20–27], details of TE
mechanism still remain unclear. It is unknown whether a nucleophilic attack of oxygen
ꢀ
Oꢁ1 of FGly on the sulfur of substrate is preceded by release of RO of sulfate
ꢀ
substrate according to a dissociative mechanism (D), scheme 2b, or release of RO is
followed by bond formation between oxygen Oꢁ1 and the sulfur of a substrate
following an associative mechanism (A) (as proposed for phosphate esters [28]),
scheme 2(a). Another possibility is simultaneous nucleophilic attack of the oxygen Oꢁ1
ꢀ
at the sulfur of the substrate and release of RO in an interchange mechanism (I).
Studies of the mechanism of uncatalyzed sulfate ester hydrolysis, presented by
Bourne et al. [29] and Hopkins et al. [30] have proved that the sulfur has mainly sulfur
tricoordination in the transition state. However, results obtained by Hengge’s group
also for uncatalyzed reaction [7] revealed that the formation of a pentacoordinated
intermediate is partially rate-determining, and the cleavage of the S–O bond takes place

Mechanistic studies of the hydrolysis of p-nitrophenyl sulfate
2475
R
#
AspC
R
(
a)
O
S
O
S
–
O
O
O–
O
O–
+
O
H (HisB)
HO
OH
O
HO
O
ROH
FGly
FGly
O
O–
S
H2O
O
HSO4–
–
O
HO
H
OH
HO
O
FGly
FGly
FGly
#
–
R
O
(b)
AspC
R
ROH
O
S
O–
O
–
O
O
S
O
O–
+
HO
OH
O
O–
H (HisB)
HO
FGly
FGly
Scheme 2. Two extreme possible reaction pathways for hydrolysis of p-NPS catalyzed by ARS from
H. pomatia. (a) An associative pathway with a five-coordinate sulfur in the transition state. (b) A dissociative
pathway with tricoordinate sulfur in the transition state.
3
4
subsequently, which is an associative mechanism. Later, on the basis of S isotope
effect on sulfate ester hydrolysis as well as theoretical considerations, Burlingham
et al. [31] proposed that cleavage of the S–O bond takes place during the rate-limiting
step, pointing to a dissociative mechanism. The results obtained for uncatalyzed
hydrolysis of sulfate ester could be helpful in explaining the reaction mechanism for
catalytic reaction; however, due to the number of conflicting conclusions it appears
not so straightforward. Therefore, more kinetic and stereochemical studies focusing
on the catalyzed reaction are required to propose the reaction mechanism.
The results obtained for sulfate ester hydrolysis catalyzed by ARS from Aspergillus
oryzae [16] indicate that the catalytic mechanism of sulfur transfer is stepwise with a
covalent sulfoenzyme intermediate. Further mechanistic considerations are based on
studies of p-nitrophenyl sulfate ( p-NPS) hydrolysis catalyzed by ARS from H. pomatia
and from Aerobacter aerogenes [22]. Their kinetic isotope effect (KIE) data obtained
for ARS from H. pomatia revealed that in the transition state both S–O bond fission
and protonation of the leaving group are far advanced, supportive for an associative or
interchange mechanism. However, the leaving group KIE data obtained for ARS from
Aerobacter aerogenes show that in the transition state protonation of the leaving
group and S–O bond fission are not simultaneous (proton transfer lags behind S–O
bond fission), indicating rather a dissociative mechanism.

2
476
I. Stawoska et al.
In spite of many kinetic and stereochemical studies, the molecular nature of the
transition state for sulfate ester hydrolysis catalyzed by ARSs is still not clear.
A useful approach to study the mechanisms of different types of reactions is
application of kinetic methods using temperature and pressure as variables. By
changing both temperature and pressure, it is possible to obtain the activation entropy
6¼
6
6¼
(
DS ), activation enthalpy (DH ), as well as volume of activation (DV ) which
allows for the characterization of the transition state/states of the studied reaction.
The pressure dependence of the equilibrium constant for binding of ligands allows
determination of reaction volumes. Both reaction volumes and activation volumes
provide unique information for determining the molecular mechanism of enzyme-
catalyzed reaction.
Here we report the results of the studies on temperature and pressure effects on
the hydrolysis of small aryl substrate, p-NPS catalyzed by ARS from H. pomatia.
The employed kinetic assay techniques (continuous UV-Vis measurements in the wide
range of temperatures as well as at ambient and elevated pressures) allow for the
extraction of the appropriate rate and activation parameters. The obtained results are
discussed in reference to the proposed reaction mechanism for hydrolysis of sulfate
esters.
2
2
. Experimental procedures
.1. Reagents
The ARS from H. pomatia was purchased from ROCHE Applied Science and the
activity of sulfatase was determined according to Baum’s procedure [32]. The protein
ꢁ
ꢀ1
was stored at 4 C and in order to get active form it was dialysed against 1 mmol L
ꢀ
1
Tris/HCl, pH 7.4 containing 0.1 mol L KCl. The enzyme was stable under such
conditions for months [23]. Other reagents such as p-nitrocatechol sulfate ( p-NCS),
potassium p-NPS, and 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) were
obtained from Sigma–Aldrich. Solutions were prepared using deionized water purified
by a Millipore system. All chemicals used throughout this study were of analytical
reagent grade.
2
.2. UV-Vis measurements
UV-Vis spectra at appropriate temperature were recorded in quartz cuvettes on a
Shimadzu UV-2100 spectrophotometer equipped with a thermostated cell compartment
ꢁ
CPS-260 (ꢂ0.1 C). A pill-box cell combined with high-pressure equipment [33] was
used to record UV-Vis spectra under elevated pressure (up to 160 MPa).
2
.3. Assay of ARS catalytic activity at ambient and elevated pressure
In all kinetic measurements, the concentration of enzyme (arylosulfatase from
ꢀ1
ꢀ9
H. pomatia) was kept constant (8 ꢃ10 mol L ), while the concentration of substrate
ꢀ
1
p-NPS) was varied from 0.15 to 4.0 mmol L . Reactions were carried out in
(

Mechanistic studies of the hydrolysis of p-nitrophenyl sulfate
parameters obtained for the hydrolysis of
p-NPS catalyzed by ARS from H. pomatia. Experimental conditions:
2477
Table 1. The Vmax and K
m
ꢀ9
ꢀ1
ꢀ3
ꢀ3
ꢀ1
[
ARS] ¼8 ꢃ10 mol L , [ p-NPS] ¼(0.15 ꢃ10
to 4 ꢃ10 ) mol L
,
ꢀ
1
ꢁ
0.1 mol L Tris/HCl, T ¼30 C.
max ꢃ10ꢀ (mmol L min
2
ꢀ1
ꢀ1
)
ꢀ1
m
K (mmol L
)
pH
V
8
8
7
7
.68
2.57 ꢂ0.72
5.42 ꢂ1.22
3.38 ꢂ0.48
2.06 ꢂ0.60
5.26 ꢂ1.58
4.36 ꢂ1.06
1.85 ꢂ0.32
1.20 ꢂ0.43
.08
.43
.08
ꢀ
1
.1 mmol L Tris/HCl buffer at pH 7.4 (or other as described in table 1 for the
0
experiments checking the pH effect on the reaction). The temperature effect on the
ꢁ
investigated reaction was studied in the temperature range 15–35 C. The reaction
was monitored spectrophotometrically by controlling p-nitrophenol ( p-NP) release at
4
the quoted errors are based on standard deviations. The pressure effect on the catalytic
57 nm. The reported rate constants are mean values from at least five kinetic runs, and
ꢁ
process was studied at a constant temperature of 30 C using a stainless steel pressure
chamber working at pressures up to 200 MPa. The reaction mixture (enzyme and
substrate at the same concentration as used for temperature dependence measurement)
was prepared at atmospheric pressure and room temperature. Experiments were
performed in Tris/HCl buffer at pH 7.4 since the pK value of Tris is relatively
a
insensitive to pressure up to 200 MPa [34]. After mixing of p-NSP with enzyme, the
solution was placed into a pill-box cell, which was placed in the pressure chamber filled
with thermostated water. The reaction mixture was equilibrated for 5–7 min prior to
measurement. The velocity of p-NP formation was determined by measuring the
absorbance changes at 457 nm. The reported rate constants are mean values from at
least three kinetic runs, and the quoted errors are based on standard deviations.
Further details of experimental conditions used in the presented experiments are
specified in figure captions.
3
3
. Results and discussion
.1. Steady state kinetics for enzyme-catalyzed hydrolysis of p-NPS
Despite the fact that the hydrolytic activity of ARSs on various types of sulfate
substrates has been studied since the early sixties, only a few results for ARS from
H. pomatia and p-NPS as its substrate have been published [22, 23, 35, 36]. However,
no pressure or temperature studies allowing for the determination of the activation
parameters for this reaction were undertaken. Since the mechanism of nucleophilic
substitution is not resolved, we decided to carry out detailed kinetic studies including
both temperature and pressure measurements. Additional information gained from
these studies could help in elucidation of the mechanism for enzymatic activity of ARSs.
In order to find the best experimental conditions for pressure measurements, the
hydrolysis of p-NPS catalyzed by ARS from H. pomatia was measured under different
pH and temperature conditions. The UV-Vis spectrum of p-NPS in a neutral buffer

2
478
I. Stawoska et al.
2
1
8
.4x10–2
1
1
0
0
0
.6
.2
.8
.4
.0
.6x10–2
.0x10–3
0.0
0
1
2
3
4
p-NPS (mM)
240
300
360
420
λ (nm)
480
540
600
Figure 1. Representative UV-Vis spectral changes recorded for hydrolysis of p-NPS catalyzed by ARS
from H. pomatia.
Experimental conditions: [ARS] ¼8 ꢃ10 mol Lꢀ1, [ p-NPS] ¼1.5 ꢃ10ꢀ4 mol L , 0.1 mol L
ꢀ
9
ꢀ1
ꢀ1
Tris/HCl
ꢁ
buffer pH 7.4, T ¼25 C.
Inset: Typical saturation plot of the reaction initial velocity (measured as the slopes of the kinetic traces
obtained from absorbance changes recorded at ꢂ ¼457 nm) as a function of substrate concentration.
exhibits a band with maximum absorbance at 282 nm. As can be seen in figure 1,
the hydrolysis of p-NPS catalyzed by ARS from H. Pomatia leads to a decrease of this
band and a new one at 400 nm, attributed to p-NP is formed. The enzyme activity was
determined from the spectrophotometrical measurements by monitoring the changes
in absorbance at ꢂ ¼457 nm which allow for the kinetic studies of all the chosen
substrate concentrations.
On the basis of the hydrolysis of various sulfate substrates [18, 21, 24, 37] the process
is characterized by Michaelis–Menten kinetics. Early theoretical considerations [38] as
well as the mechanism of sulfatases activity proposed by Lukatela et al. [14] indicate
that hydrolysis of small aryl sulfates can be described by a simple model, equation (3):
k1
k2
E þS ꢀ!ES ꢀ!E þY
ð3Þ
ꢀ
kꢀ1
where k and k are the rate constants for formation and dissociation of the enzyme-
ꢀ1
1
substrate complex, respectively (Kd ¼kꢀ1=k1 is the dissociation constant), while k is
2
the rate constant for formation of the reaction product Y. Using the King–Altman
method [39], assuming that reaction (3) is a cyclic process, the overall velocity equation
can be written as follows:
dY
k1k2½S ꢄ
v ¼dt
¼
½E ꢄ
ð4Þ
kꢀ1 þk2 þk1½S ꢄ
If the majority of the active sites of the enzyme are saturated by the substrate,
k2 equals kcat and the maximal velocity Vmax is defined as kcat[E ], where [E ] denotes
t
t
the total enzyme concentration. By defining the Michaelis constant as in equation (5):
k2 þkꢀ1
Km ¼
ð5Þ
k1

Mechanistic studies of the hydrolysis of p-nitrophenyl sulfate
2479
Table 2. Catalytic properties of selected ARSs from various organisms.
ꢀ1
Enzyme
Substrate pH optimum
K
m
(mmol L
)
References
ARS A, human
p-NCS
5.5
.5
0.4
4.2
.5
0.4
2
8
1.09
0.102
2.3
0.19–1.79
0.03–1.25
0.824
0.47
[43]
[45]
[21]
[41]
[44]
4
3
ARS C, human
ARS C from Ox liver
p-NPS
p-NPS
p-NCS
p-NPS
7.0
7.9–8
7.5
7.3–7.5
ARS from digestive juice of H. pomatia
[23]
[22]
ꢅ5
p-NCS
p-NPS
p-NCS
p-NPS
p-NPS
p–NCS
7.2
6–7.3
ARS from digestive juice/gland of H. pomatia
[35]
6.5–7.5
ARS from A. aerogenes
ARS from Alcaligenes metalcaligenes
7.0
8.8
8.0
[22]
[42]
0.22
the velocity equation derived from equation (4) can be converted to:
Vmax
v ¼
Km
ð6Þ
þ1
½
S ꢄ
For each investigated substrate concentration, the active sites of the enzyme were
saturated as confirmed by the observation of the saturation plot of initial velocity versus
p-NPS concentration (inset of figure 1). These findings give rise to the calculation of Km
and Vmax parameters according to the standard procedure [40].
3.2. The pH influence on the enzymatic activity of ARS from H. pomatia
Examination of the influence of pH on p-NPS hydrolysis catalyzed by ARS from
H. pomatia has shown a relatively strong effect of pH on maximal velocity of this
reaction even in the narrow pH range (8.68–7.08), as described in table 1. The highest
velocity can be obtained at pH 7.8–7.9, a little higher than values of optimum pH
reported for other ARSs from H. pomatia (table 2). The differences are probably caused
by variations in experimental conditions (e.g. temperature, buffer content) as well as
in a source of the protein. Moreover, it is suggested that the optimum pH varies
appreciably with the concentration of substrates [35] and enzyme [26]. For all
types of ARSs, optimum pH for hydrolysis of p-NCS was lower than for p-NPS [21–23,
3
5, 41–45].
Studies of pH influence on catalytic parameters of p-NPS hydrolysis indicate that Km
increases with pH, which is in good agreement with previously published data [22, 23],
whereas Vmax increases with pH up to 7.9 (the optimum pH) followed by a rapid
decrease (table 1). According to previous analysis [22], the dependence of log(Vmax/Km)
versus pH has clearly bell-shaped profile (figure 2). Fitting these data to equation (7),
Vmax
C
log
¼log
ð7Þ
þ
K
1 þ½
H ꢄ
þ
a2
þ
Km
Ka
½H ꢄ
1

2
480
I. Stawoska et al.
–
–
–
–
1.8
2.0
2.2
2.4
7.2
7.6
8.0
pH
8.4
8.8
Figure 2. The bell-shaped pH rate profile for hydrolysis of p-NPS catalyzed by ARS from H. pomatia.
ꢀ9
ꢀ1
ꢀ3
ꢀ3
ꢀ1
ꢀ1
Experimental conditions: [ARS] ¼8 ꢃ10 mol L , [ p-NPS] ¼(0.15 ꢃ10 to 4 ꢃ10 mol L , 0.1 mol L
ꢁ
Tris/HCl buffer, T ¼30 C.
Table 3. Kinetic parameters for the hydrolysis of p-NPS catalyzed by
ꢀ1
ꢀ
9
ARS from H. pomatia. Experimental conditions: [ARS] ¼8 ꢃ10 mol L
,
ꢀ
3
ꢀ1
ꢀ1
[
p-NPS] ¼(0.15 ꢃ10 to 4 ꢃ10ꢀ3) mol L , 0.1 mol L Tris/HCl buffer pH 7.4.
ꢀ
2
3
Pressure
(MPa)
Temperature
(K)
V
(mmol L min
max ꢃ10
K
(mmol L )
m
k
(min
catꢃ10
ꢀ1
ꢀ
1
ꢀ1
ꢀ1
)
)
0
.1
288
2.06 ꢂ0.20
2.01 ꢂ0.49
2.21 ꢂ0.63
2.79 ꢂ0.54
2.71 ꢂ0.26
3.58 ꢂ0.25
4.36 ꢂ0.11
2.32 ꢂ0.70
2.23 ꢂ1.05
2.47 ꢂ1.17
1.95 ꢂ0.62
2.04 ꢂ0.91
2.47 ꢂ0.18
2.49 ꢂ0.52
2.01 ꢂ0.47
2.31 ꢂ0.32
1.52 ꢂ0.11
2.12 ꢂ0.12
2.50 ꢂ0.55
1.60 ꢂ0.40
1.61 ꢂ0.63
1.62 ꢂ0.64
1.12 ꢂ0.27
0.82 ꢂ0.25
2.57 ꢂ0.25
2.51 ꢂ0.61
2.77 ꢂ0.78
3.48 ꢂ0.67
3.38 ꢂ0.32
4.47 ꢂ0.31
5.45 ꢂ1.41
2.90 ꢂ0.87
2.79 ꢂ1.31
3.08 ꢂ1.46
2.43 ꢂ0.77
2.55 ꢂ1.14
2
2
2
2
3
3
90.5
93
95.5
98
03
08
5
0
0
20
60
303
4
8
1
1
can estimate pKa1 (acidic limb) and pKa2 (alkaline limb), equal 6.74 ꢂ0.28 and
8
suggest that deprotonation of one diol group of the enzyme ( pKa ) as well as
.04 ꢂ0.01, respectively. Our results together with those obtained by Gibby et al. [22]
1
protonation of the leaving group ( pKa ) (scheme 2) occurs in the system.
2
3
.3. Temperature effect on K and Vmax
m
The kinetic behavior of p-NPS hydrolysis in buffered solution (pH 7.4) catalyzed by
ꢁ
H. pomatia was studied in the temperature range 15–35 C. Systematic measurements
of the maximal velocity, Vmax, reveal the general tendency of this parameter to increase
with temperature (table 3). Thus, the catalytic rate, kcat, increases in a temperature
ꢁ
range 15–35 C by a factor of two. A slightly different behavior was observed for K as
m

Mechanistic studies of the hydrolysis of p-nitrophenyl sulfate
2481
6
4
3
1
0
.0
.5
.0
.5
.0
–
1.5
2
85
290
295
300
305
310
T (K)
Figure 3. Temperature effect on the Michaelis constant, K
m
, for H. pomatia ARS-catalyzed hydrolysis of
ꢀ
9
ꢀ1
ꢀ3
ꢀ3
ꢀ1
,
p-NPS. Experimental conditions: [ARS] ¼8 ꢃ10 mol L , [ p-NPS] ¼(0.15 ꢃ10
to 4 ꢃ10 ) mol L
ꢀ1
ꢁ
0
.1 mol L Tris/HCl buffer pH 7.4, 15–35 C.
illustrated in figure 3. Since K is characterized by equation (5), it is possible to consider
m
two cases: (1) if k2 ꢆkꢀ1, the dissociation of ES complex back to E þS (equation 3)
will be much faster than product formation and we can make an assumption that:
k2 þkꢀ1
k1
kꢀ1
k1
Km ¼
ffi
ffiKd
ð8Þ
where K is the dissociation constant of the ES complex. Taking into account that V
d
max
ꢁ
ꢁ
increases with temperature, decrease of Km in a temperature range from 15 C to 25 C
suggests that k has a minor contribution to K . Thus, under these conditions K is a
measure of the affinity of the enzyme for the substrate; (2) if k is comparable to k , it is
2
m
m
2
–1
not possible to neglect k , as well as its participations in Km value. The increase of the
2
ꢁ
K value in a temperature range 25–35 C suggests faster product formation comparing
m
to slower regeneration of the substrate.
In order to characterize the transition state, the activation parameters DH and DS
were calculated according to the Eyring equation:
6
6
ꢀ
ꢁ
ꢀ
ꢁ
ꢀ
ꢁ
6
6
6
k T
B
ꢀDG
k T
B
ꢀDH
DS
k ¼
exp
¼
exp
exp
ð9Þ
h
RT
h
RT
R
Catalytic rate constant, kcat, was used to generate an Eyring plot of lnðkcat=T Þversus
1/T (figure 4) from which activation parameters were found and summarized in table 4.
The parameters obtained for enzymatic hydrolysis of p-NPS are characterized by a
6
ꢀ1
positive activation enthalpy, DH ¼28 ꢂ2 kJ mol and a negative value of activation
6¼
ꢀ1 ꢀ1
entropy, DS ¼–83 ꢂ10 J mol
K . The negative value of activation entropy suggests
6¼
association or interchange rather than dissociation mechanism. Determination of DS
6
in principle involves an extrapolation to 1/T, which leads to differences in the DS
values between different experiments apparently much larger than indicated by
standard deviations for individual experiments. This discrepancy was the motivating
factor for measuring the influence of pressure on the tested system.

2
482
I. Stawoska et al.
4.0
3.2
2.4
1.6
3
.3x10–3
3.4x10–3
/T (K–1
3.4x10–3
1
)
Figure 4. Eyring plot of lnðkcat=TÞvs. 1/T for hydrolysis of p-NPS catalyzed by ARS from H. pomatia.
ꢀ1
ꢀ9
ꢀ1
ꢀ3
ꢀ3
ꢀ1
Experimental conditions: [ARS] ¼8 ꢃ10 mol L , [p-NPS] ¼(0.15 ꢃ10 to 4 ꢃ10 ) mol L , 0.1 mol L
Tris/HCl buffer pH 7.4.
Table 4. Kinetic and volumetric parameters for the hydrolysis
of p-NPS catalyzed by ARS from H. pomatia.
Parameters
Values
3
ꢀ1
kcat ꢃ10 ðmin
Þ
2.90 ꢂ0.87
28.04 ꢂ2.26
ꢀ83.06 ꢂ9.62
2.5 ꢂ1.6
6
ꢀ1
DH ðkJ mol
Þ
6
ꢀ1
ꢀ1
DS ðJ K mol
Þ
DV ðcm molꢀ1Þ
6
3
cat
K
m
independent of compressibility
3
ꢀ1
ꢀ1
DVbð1Þðcm mol
Þ
ꢀ1
0
3
DVbð2Þðcm mol
Þ
21.2 ꢂ0.8
6
3
a
b
DV ðAÞðcm mol
Þ
(ꢀ18.7 Ä2.5 )
obs
m
K dependent on compressibility
3
ꢀ1
DVbð3Þðcm mol
Þ
8.4 ꢂ5.1
6
3
ꢀ1
a
b
DV ðBÞðcm mol
Þ
(ꢀ5.9 Ä2.5 )
obs
DꢃKm ðcm mol MPaꢀ1Þ
3
ꢀ1
ꢀ0.23 ꢂ0.06
a
Calculated for ½SꢄꢆKm.
b
Calculated for ½SꢄꢇKm.
3.4. Determination of binding and activation volumes
Studies of enzyme-catalyzed hydrolysis of p-NPS under elevated pressure show
dependence of the steady-state catalytic parameters, i.e. K , Vmax, and kcat on pressure.
m
All data are presented in table 4. The pressure effect on catalytic parameters is described
by equations (10) and (11):
ꢀ
ꢁ
@
ln Km
DVb
RT
¼
ꢀ
ð10Þ
ð11Þ
@
P
T
ꢀ
ꢁ
6¼
cat
@
ln kcat
DV
¼
ꢀ
RT
@
P
T

Mechanistic studies of the hydrolysis of p-nitrophenyl sulfate
2483
In equation (10), DVb is the volume change upon productive substrate binding. Since
Km can be regarded as a dissociation constant for productive bound substrate (b),
6
DV ¼ꢀDVK [46]. The DV in equation (11) is the activation volume of the catalytic
b
m
cat
3 ꢀ1 ꢀ1
step. R denotes the gas constant (8.314 cm MPa mol K ) and T is the absolute
temperature. The pressure dependence of K and k can be described either by linear
m
cat
equations (12a) and (13a), respectively, or quadratic equations (12b) and (13b),
respectively, which include compressibility effect [46].
DVKm
RT
ln Km ¼ln Km,P ꢀ
P
ð12aÞ
ð12bÞ
0
DVKm
DꢃKm
2RT
2
ln Km ¼ln Km,P ꢀ
P þ
P
0
RT
6¼
DV
cat
ln kcat ¼ln k0,P0
ꢀ
P
ð13aÞ
ð13bÞ
RT
6
6
DV
Dꢃ
2RT
cat
cat
2
ln kcat ¼ln k0,P ꢀ
P þ
P
0
RT
6¼
cat
where Dꢃ is the activation compressibility and DꢃKm is the compressibility changes
upon substrate binding.
Applying the Michaelis–Menten model described by equation (4) and differentiation
of the rate equation with respect to pressure give an expression for observed activation
6¼
obs
volume (DV ) [46]:
DVb
6
6¼
kcat
DV ¼DV
ꢀ
ð14Þ
obs
½S ꢄ
1
þ
Km
Equation 14 at two boundary conditions gives: for ½Sꢄ 55 Km, observed activation
6
6
6
volume can be calculated as DV ¼DV ꢀDVb while for ½Sꢄ44Km, DV
cat
is
obs
kcat
obs
6
dominated by a contribution of DV . In the first approximation, the binding and
k
activation volumes can be easily found from equations (12a) and (13a), respectively.
However, there are at least three factors which influence linear dependence such as
changes in: (1) quaternary structure and/or modification of the active site architecture,
(2) a compressibility and (3) the rate-determining step which can occur under elevated
pressure [46]. We have found that in the tested system, the dependence of ln k
versus pressure is linear (figure 5), allowing us to define Dꢃ as zero for this enzyme.
cat
6¼
cat
From equation (13a), we find that the catalytic activation volume has a value
3
ꢀ1
2
.5 ꢂ1.6 cm mol
.
The obtained data for pressure effect on K can be discussed in two separate ways,
m
since dependence of ln Km versus pressure allows applying either linear (equation 12a)
or quadratic equations (equation 12b). If we presume that Km is independent of
compressibility, DꢃKm ¼0 and the linear plots can be fitted (figure 6a). Since the
pressure dependence of Km is biphasic (figure 6a), the obtained data should be
considered separately in two pressure ranges, ambient to 80 MPa, DV ð1Þ, and from
b
8
0 to 160 MPa, DVbð2Þ. The substrate binding to the active site seems not influenced
by pressure lower than 80 MPa, for which DV ð1Þffi0. Consequently, the observed
b
6
6¼
3
ꢀ1
activation volume DV ðA1Þ¼DV and equals 2.5 ꢂ1.6 cm mol . Decrease of Km
obs
kcat

2
484
I. Stawoska et al.
1
0
9
8
7
6
0
40
80
120
160
P (MPa)
Figure 5. Plot of ln(kcat) vs. pressure for p-NPS hydrolysis catalyzed by ARS from H. pomatia.
Experimental conditions: [ARS] ¼8 ꢃ10 mol L , [ p-NPS] ¼(0.15 ꢃ10ꢀ3 to 4 ꢃ10ꢀ3) mol L , 0.1 mol L
ꢀ
9
ꢀ1
ꢀ1
ꢀ1
ꢁ
Tris/HCl buffer pH 7.4. Pressure range 5–160 MPa, T ¼30 C.
(a)
(
b)
2
1
0
1
2
2
1
0
–
–
–1
–2
0
40
80
120
160
0
40
80
120
160
P (MPa)
P (MPa)
Figure 6. The dependence of ln K
without (a) and with (b) the influence of compressibility changes upon substrate binding.
m
vs. pressure for p-NPS hydrolysis catalyzed by ARS from H. pomatia
ꢀ
9
ꢀ1
ꢀ3
ꢀ3
ꢀ1
ꢀ1
Experimental conditions: [ARS] ¼8 ꢃ10 mol L , [ p-NPS] ¼(0.15 ꢃ10 to 4 ꢃ10 ) mol L , 0.1 mol L
ꢁ
Tris/HCl buffer pH 7.4. Pressure range 5–160 MPa, T ¼30 C.
3
ꢀ1
in a pressure range 80–160 MPa allowed us to calculate DVbð2Þ¼21.2 ꢂ0.8 cm mol
.
In order to determine activation volume in this pressure range, it was necessary to take
into account substrate concentration. For ½SꢄꢆK , the minimal value of the activation
m
6
3
ꢀ1
volume can be found as DV ðA2minÞ¼ꢀ18.7 cm mol , whereas for ½SꢄꢇKm,
obs
obs
3
ꢀ1
observed activation volume DV ðA2 Þ¼2.5 ꢂ0.8 cm mol (table 4). Therefore,
max
assuming independence of Km from compressibility for the broad range of substrate
6
concentration, the calculated values of the observed activation volume (DV ðAÞ) varies
obs
3
ꢀ1
.
from ꢀ18.7 to 2.5 cm mol
Fitting a quadratic equation (12b) to the same data (figure 6b) allows for the
3
calculation of DV (3), which was found to be 8.4 ꢂ5.1 cm mol as well as DꢃKm with
ꢀ1
b

Mechanistic studies of the hydrolysis of p-nitrophenyl sulfate
2485
3
ꢀ1
ꢀ1
value of ꢀ0.23 ꢂ0.06 cm mol MPa (table 4). Considering a different substrate
6
concentrations (see above), we calculated the observed activation volume DV ðBÞ
obs
3
ꢀ1
ranging from ꢀ5.9 to 2.5 cm mol for ½SꢄꢆKm and ½SꢄꢇKm, respectively. The
decrease of activity may be an effect of modifications of enzyme conformation or
changes of the substrate binding mechanism [47, 48].
Volume changes upon substrate binding (DV ) both models (K independent and
b
m
dependent on compressibility) are positive and some contribution to these values
can originate from desolvation of substrate entering the active site [14]. Generally,
the changes of activation volumes were negative for low substrate concentrations
and close to zero (positive) for high substrate concentrations (table 4). The
interpretation of obtained values is often ambiguous because experimental values are
a sum of three components, as seen in equation (15).
DV ¼DV þDV¼ þDV¼
6
6
6
6
ð15Þ
intr
solv
canf
6¼
where DV the intrinsic contribution, refers to formation and disruption of chemical
intr
6¼
bonds, DV
is associated with the influence of water molecules on surrounding
shows the influence of conformational changes as a result of
solv
6
moieties, and DV
canf
chemical reactions. Therefore, it is necessary to distinguish these three components
in the obtained activation volume. As reported previously, solvation contribution for
enzymatic reaction is usually dominant for overall activation volume [49]. Also in this
case, water seems to play a key role, especially because it is engaged in the mechanism
6
of enzymatic hydrolysis of p-NPS (scheme 2). DV depends on the character of bonds
which are formed or broken during the reaction steps. For disruption of ester bonds this
intr
3
ꢀ1
value was found to be around þ10 cm mol [50]. The conformational contribution can
usually be neglected, with the exception of oligomeric enzymes. Thus, if we assume
independence of the system from compressibility changes upon substrate binding,
6
3
ꢀ1
we can calculate the solvation effect in a range of DV ð1Þ¼ꢀ7.5 cm mol for the
solv
solv
6
3
ꢀ1
pressure up to 80 MPa and DV ð2Þfrom ꢀ7.5 to ꢀ28.7 cm mol at higher values
of pressure. Accounting for the compressibility influence on Km in the tested pressure
3
ꢀ1
range gives us the solvation contribution form ꢀ15.9 to ꢀ8.5 cm mol . All the
3
ꢀ1
obtained values are negative in the range from ca ꢀ29 to ꢀ8 cm mol and in well
agreement with values obtained for other enzymes [51].
4
. Conclusions
6¼
We have obtained negative DS , which could indicate the associative or associative
interchange mechanism for hydrolysis of sulfate ester. The value of the observed
6
activation volume (DV ), independent of whether it was calculated with an
assumption that Km depends on compressibility changes upon substrate binding or
obs
3
not, were found to be from close to zero positive (2.5 cm mol ) to negative
ꢀ1
3
ꢀ1
(
(
ꢀ18.7 cm mol ). The small positive value of catalytic activation volume
3
ꢀ1
2.5 ꢂ1.6 cm mol ) and rather big uncertainty suggest that pressure only slightly
affects catalytic reaction rate. Based on both temperature and pressure influence on
catalytic reaction rate (kcat) and Michaelis constant (K ), we suggest the associative
interchange mechanism. In the transition state in which sulfur from sulfate substrate
m

2
486
I. Stawoska et al.
has the character of five-coordinative species. The bond that is formed between
incoming nucleophile and the sulfur is expected to be as strong as a bond between sulfur
and the leaving groups.
In these studies, for the first time, activation parameters of p-NPS hydrolysis
catalyzed by ARS from H. pomatia were determined. Results of kinetic measurements
showed the influence of pressure on the mechanism. We discussed two separate ways
of interpretation of the obtained results, which were already presented for different
enzymatic systems [51, 52]. Considering independence of Km from compressibility
changes upon substrate binding, we suggest that in a pressure range up to 80 MPa,
the system is characterized by an interchange mechanism of nucleophilic substitution.
6
The slightly positive value of DV
implies that the sulfur in a transient product
obs
is three- rather than five-coordinate. Nevertheless, increase of pressure shifts this
dependence to associative mechanism and strongly indicates a five-coordinate transient
product. Taking into account the influence of DꢃKm , we found confirmation of our
conclusions concerning the type of mechanism of p-NPS hydrolysis catalyzed by ARS
from H. pomatia.
Acknowledgment
The authors are grateful for the partial financial support from COST Chemistry
Action D30.
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3