A Nucleophilic Catalysis Step is Involved in the Hydrolysis of
Aryl Phosphate Monoesters by Human CT Acylphosphatase*
Paolo
Paoli,
Luigia
Pazzagli,
Elisa
Giannoni,
Anna
Caselli,
Giampaolo
Manao,
Guido
Camici
, and
Giampietro
Ramponi
From the Department of Biochemical Sciences, University of
Florence, Viale Morgagni 50, 50134 Florence, Italy
Received for publication, July 11, 2002, and in revised form, October 28, 2002
 |
ABSTRACT |
Acylphosphatase, one of the smallest enzymes, is
expressed in all organisms. It displays hydrolytic activity on acyl
phosphates, nucleoside di- and triphosphates, aryl phosphate
monoesters, and polynucleotides, with acyl phosphates being the
most specific substrates in vitro. The mechanism of
catalysis for human acylphosphatase (the organ-common type isoenzyme)
was investigated using both aryl phosphate monoesters and acyl
phosphates as substrates. The enzyme is able to catalyze
phosphotransfer from p-nitrophenyl phosphate to glycerol
(but not from benzoyl phosphate to glycerol), as well as the inorganic
phosphate-H218O oxygen exchange reaction in the
absence of carboxylic acids or phenols. In short, our findings point to
two different catalytic pathways for aryl phosphate monoesters and acyl
phosphates. In particular, in the aryl phosphate monoester hydrolysis
pathway, an enzyme-phosphate covalent intermediate is formed, whereas
the hydrolysis of acyl phosphates seems a more simple process in which the Michaelis complex is attacked directly by a water molecule generating the reaction products. The formation of an enzyme-phosphate covalent complex is consistent with the experiments of isotope exchange
and transphosphorylation from substrates to glycerol, as well as with
the measurements of the Brønsted free energy relationships using a
panel of aryl phosphates with different structures. His-25 involvement in the formation of the enzyme-phosphate covalent complex
during the hydrolysis of aryl phosphate monoesters finds significant
confirmation in experiments performed with the H25Q mutated enzyme.
 |
INTRODUCTION |
Acylphosphatase (ACP),1
one of the smallest enzymes (11 kDa), is expressed in all known
organisms, and its cellular function has not been hitherto fully
understood. Several reports indicate that ACP is involved in
controlling membrane ion pumps (Refs. 1 and 2 and citations therein)
because in vitro it displays hydrolytic activity against the
aspartyl phosphate intermediate formed during the action of membrane
Na+,K+- and Ca2+-ATPases. The fact
that ACP is able to bind sarco-/endoplasmic reticulum calcium ATPase
(3) is in line with the preceding hypothesis. Other reports note the
involvement of ACP in cell differentiation and apoptosis (4-6); the
ACP level is greatly enhanced during the differentiation process that a
number of specific compounds (triiodothyronine, phorbol 12-myristate
13-acetate, aphidicolin, and hemin) induce, and moreover, the enzyme is
able to migrate into the nucleus during both differentiation and
apoptosis (7, 8). The overexpression of ACP in yeast cells enhances the
rate of glycolysis, suggesting that it is capable of hydrolyzing 1,3-bisphosphoglycerate in vivo (9).
Site-directed mutagenesis experiments suggest that Arg-23 and Asn-41
are essential residues (10, 11), Arg-23 being involved in the binding
of the substrate phosphate moiety (12). A model based on x-ray
crystallography data for the catalytic hydrolysis of acyl phosphates by
ACP was previously proposed by Thunnissen et al. (12).
In this article we demonstrate that human CT acylphosphatase catalyzes
the hydrolysis of aryl phosphates displaying a different mechanism from
the one it uses for acyl phosphate hydrolysis. For
p-nitrophenyl phosphate hydrolysis, we found that the enzyme uses His-25 to perform a nucleophilic catalysis step, which, by contrast, it does not use in the catalytic hydrolysis of acyl phosphates.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
Benzoyl phosphate was synthesized as
described previously (13). The aryl phosphate monoesters 4-cyano and
3-chloro were synthesized as dicycloexylammonium salts in accordance
with Zhang and Van Etten (14), whereas p-nitrophenyl
phosphate, phenyl phosphate,
-naphtyl phosphate, and
L-tyrosine phosphate where purchased from Sigma.
[18O]water at 97% isotope enrichment was purchased from
Cambridge Isotope Laboratories. All other reagents were the purest
commercially available grade.
Enzymes--
wild-type human CT-ACP was produced and purified as
previously described (15). The H25Q mutant of CT-ACP was obtained by oligonucleotide-directed mutagenesis using a unique site elimination (USE) mutagenesis kit based on the USE method developed by Deng and
Nickoloff (16). The mutations were confirmed by DNA sequencing according to Sanger et al. (17) and by amino acid analysis
of the purified proteins.
Determination of Enzyme Activity--
Benzoylphosphatase
activity was assayed using a continuous optical test in accordance with
Ramponi et al. (18). The hydrolytic activity on aryl
phosphates was in general assayed as follows: substrates were dissolved
in 1 ml of 0.1 M acetate buffer, pH 5.3, containing 1 mM EDTA (Buffer 1). The mixture was incubated at 37 °C
and after the addition of the enzyme, aliquots were taken at different
times to assay the released Pi by the method of Baginski et al. (19). The p-nitrophenyl phosphatase
activity was assayed at 37 °C; pNPP was dissolved in Buffer 1 at the
final volume of 1 ml. The reaction was stopped by the addition of 4 ml
of 0.1 M KOH, and the released p-nitrophenolate
ion was measured at 400 nm (
= 18,000 M
1cm
1).
Inorganic Phosphate-Water Medium 18O
Exchange--
An orthophosphoric acid solution (100 mM
final concentration) was adjusted to pH 5.3 with NaOH. Then, 25 µl
were withdrawn and transferred to a small screw-cap conical vial and
dried. The residue was dissolved in 25 µl of
[18O]water, and 1 µl of human CT-ACP (0.28 g/liter) was
added. The mixture was incubated at room temperature. Aliquots of 2 µl were withdrawn at varying incubation times and diluted with water
to 100 µl in order to perform MS analyses. To verify the performance of the method, a parallel experiment with calf intestine alkaline phosphatase was carried out (in this experiment, the pH of the incubation mixture (25 µl) was adjusted to 9.0, and 0.14 units of
alkaline phosphatase were added (the unit is defined as the amount of
enzyme that catalyzes the hydrolysis of 1 µmol of pNPP at 25 °C
and pH 9.0)).
Mass Spectrometry--
Electrospray mass spectra in negative ion
mode were obtained under the following conditions: ion spray voltage,
2.2 kV; nozzle potential, 120 V; quadrupole DC potential, 8.5 V;
quadrupole rf potential, 350 V. Analyzer settings: push pulse
potential, 770 V; pull pulse potential, 250 V; pull bias potential,
11.5 V; acceleration potential, 4 kV; reflector potential, 1.5 kV;
detector potential, 2.05 kV. Scan range m/z,
60-300; scan rate, 8 ms/atomic mass.
 |
RESULTS |
Human CT-ACP Catalyzes the Transfer of Phosphate from pNPP to
Glycerol but Not from Benzoyl Phosphate--
The ability of ACP to
catalyze phosphate transfer was verified by partition experiments using
two different substrates, pNPP and benzoyl phosphate. Wild-type enzyme
was added to Buffer 1 solution containing pNPP or, alternatively,
benzoyl phosphate either in the presence or absence of 1 M
glycerol, a nucleophile that may compete with water in attacking the
phosphorus atom of the hypothesized E-P covalent
intermediate (see Scheme 1).
At the appropriate times, aliquots of the mixtures were drawn off,
and measurements were taken of the quantities of Pi and pNP
or Pi and benzoate released. The overall rates of substrate turnovers (hydrolysis plus substrate transfer) were measured by the
quantity of pNP or benzoate (from pNPP or benzoyl phosphate, respectively) produced, whereas the hydrolysis rates were measured by
the quantity of inorganic phosphate produced. Table
I shows that, in the absence of glycerol,
the hydrolysis rates of pNPP or benzoyl phosphate, determined by the
pNP or benzoate assays, were very close to those determined by the
phosphate assays. In the presence of 1 M glycerol, we
observed an increase in pNPP but not of benzoyl phosphate overall
turnover. Table I shows the molar ratios of pNP/Pi produced
by the ACP catalysis; at pH 5.3 and 6.5 they were, respectively, 0.97 and 0.94 in water, and 1.77 and 1.97 in 1 M glycerol. Table
I also shows the results of experiments carried out with benzoyl
phosphate; at pH 5.3, we found the benzoate/Pi molar ratio
in water to be 1.01 and, in the water-glycerol mixture, 0.96, whereas
at pH 6.5, we found it to be 1.0 in water and 0.97 in 1 M
glycerol.
View this table:
[in this window]
[in a new window]
|
Table I
Partition experiments with wild-type and H25Q mutant of CT
acylphosphatase
Experiments were performed at 37 °C in 0.1 M sodium
acetate buffer, pH 5.3, and 0.1 M sodium cacodylate buffer,
pH 6.5. Reactions were started by adding the appropriate amount of
enzyme. At varying times, aliquots were withdrawn to assay,
respectively, the released Pi and p NP for pNPP experiments and
Pi and benzoate for benzoyl phosphate experiments. R-OH
indicates p-nitrophenol or benzoate released from pNPP or
benzoyl phosphate (B-P), respectively. wt, wild type. Experiments were
performed with 2 mM B-P and 10 mM pNPP.
|
|
Glycerol Interacts with and Activates ACP--
On measuring the
fluorescence emission spectra of ACP at varying concentration of
glycerol, we found that a higher glycerol concentration causes both a
small drop of the signal and a small red-shift of the tryptophan
emission band (Fig. 1A).
F increases hyperbolically in the range 0-0.2
M glycerol, suggesting that ACP binds glycerol. In the
range 0-0.32 M glycerol, data fit well to the following
equation (Fig. 1B),

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
The effects of glycerol concentration on the
fluorescence emission of human CT-ACP and on its main kinetic
parameters. A, fluorescence emission spectra of human
CT-ACP in the absence and presence of glycerol. Spectra were acquired
with a Shimadzu model RF-5000 spectrofluorimeter; the excitation
wavelength was 280 nm. Continuous line, without glycerol;
dashed line, with 0.3 M glycerol. The enzyme was
dissolved in 0.1 M acetate buffer, pH 5.3, containing 0.15 M NaCl. In B, the experimental points indicate
the relative difference of the fluorescence emission signal at 335 nm
as a function of glycerol concentration. C, plot of
kcat/Km versus
glycerol concentration; D, plot of
kcat versus glycerol concentration.
The initial rates of pNPP hydrolysis at the indicated glycerol
concentrations were determined at pH 5.3 and 37 °C.
|
|
|
(Eq. 1)
|
where KG is the dissociation constant of
the enzyme-glycerol complex,
Fmax is the
maximum value of
F, and Gly-OH indicates glycerol. This
enables us to calculate a KG value of 17 ± 1 mM.
The plot of kcat/Km against
glycerol concentration (Fig. 1C) also shows hyperbolic
behavior. Data fitting to the following equation (Fig.
1C),
|
(Eq. 2)
|
where K' is the size of the hyperbolic increase of
kcat/Km due to glycerol
binding and KG indicates the dissociation constant of the enzyme-glycerol complex, gives
KG = 15 ± 4 mM, a value close
to that calculated from fluorescence.
To assess the effect of glycerol on kcat, we
derived the overall kcat of pNPP turnover from
Scheme 1 (20, 21) as follows.
|
(Eq. 3)
|
When the rate-limiting step is the hydrolysis or alcoholysis of
E-P, Equation 3 can be reduced as follows.
|
(Eq. 4)
|
Although the model described in Equation 4 indicates a linear
correlation between kcat and the concentration
of glycerol, Fig. 1D shows that the data agree with a
biphasic increase of kcat, the initial phase
(0-0.2 M glycerol) being hyperbolic and the secondary
linear. Thus, the data fit Equation 5 quite well,
|
(Eq. 5)
|
where k" is the size of the hyperbolic increase of
kcat caused by glycerol binding,
KG is the dissociation constant of the enzyme-glycerol complex, and k4 is the rate
constant for alcoholysis of E-P (Scheme 1). From the
kcat data we calculate a
KG of 74 ± 15 mM, a value that
is about 5-fold higher than that calculated from the data of
fluorescence and kcat/Km.
Considering that kcat/Km is
the apparent second-order constant for the reaction of free enzyme and
free substrate and that kcat is the first order
kinetic constant for the hydrolysis of the E-P covalent
complex, the difference in the value of KG
indicates that the free enzyme binds glycerol more strongly than the
E-P complex does.
The second-order rate constant (k4) for
phosphotransfer and the E-P hydrolysis constant
(k3) can be determined from Equation 5 (Fig.
1D). We found that k4 = 0.10 ± 0.01 M
1s
1 and
k3 = 0.11 ± 0.01 s
1.
The ratio of the rate of phosphotransfer (k4) to
the rate of hydrolysis (k3' = k3/[H2O] (Scheme 1) is 50.5. This
value, compared with those of other phosphatases forming E-P
covalent intermediates in their catalytic pathways (21), indicates a
moderate tendency of ACP to catalyze phosphotransfer.
The results of the fluorimetry experiments performed at low glycerol
concentration demonstrate that glycerol interacts with the enzyme
causing a hyperbolic increase of the overall activity. Nevertheless, at
glycerol concentrations above 0.2 M, the linear increase of
activity agrees with the fact that alcoholysis competes with hydrolysis
for the E-P breakdown (Scheme 1).
Inorganic Phosphate-H218O Oxygen Exchange
Experiments--
Enzyme-catalyzed 18O exchange between
Pi and water was observed in a number of enzyme such as
acid and alkaline phosphatases, ATPases, low Mr
phosphotyrosine protein phosphatase, and the catalytic domain of
leukocyte antigen-related phosphotyrosine protein phosphatase (Ref. 22
and citations therein). All of these enzymes indeed form a
phosphoenzyme in their pathways. 18O exchange
is therefore a convenient technique for tracing E-P covalent
complex formation in an enzymatic pathway. Human CT-ACP is able to
catalyze the inorganic phosphate-H218O oxygen
exchange reaction (Fig. 2). Experiments
were performed at 25 °C and pH 5.3. MS spectra of control samples to
which no enzyme was added confirm that the spontaneous exchange
reaction was negligible (Fig. 2E). Fig. 2, A-C,
shows the MS spectra of the mixtures containing wild-type ACP measured
at 25, 49, and 76 h and demonstrates that the enzyme is able to
catalyze the time-dependent incorporation of
18O atoms into phosphate when incubated with unlabeled
Pi and H218O in the absence of
p-nitrophenol. All possible isotopomers are clearly present
after 76 h of incubation. Taking into account the preceding
results, we deduce that, in the presence of the enzyme and in the
absence of phenols, the inorganic
phosphate-H218O oxygen exchange can occur only
if an E-P covalent intermediate is formed and then
hydrolyzed by cycling the two terminal steps in the overall catalytic
process shown in Scheme 2.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 2.
Electrospray MS-spectra from inorganic
phosphate-H218O oxygen exchange
experiments. The panels show the intensities of ions relative to
that of the H2P16O4
isotopomer, for which intensity was fixed at 100%. The incubation
times are indicated in the panels. wt, wild type.
|
|

View larger version (5K):
[in this window]
[in a new window]
|
Scheme 2.
The overall catalytic pathway of aryl
phosphate monoester hydrolysis by human CT-ACP. E, enzyme;
Ar, aryl; P, phosphate group.
|
|
Fig. 2F illustrates the MS spectrum of an experiment
performed with alkaline phosphatase, an enzyme that forms an
E-P covalent intermediate in its catalytic pathway (23).
This experiment, which shows the rapid incorporation of all oxygen
isotopomers into phosphate, is a good control on the performance of
the technique used here.
Structure-Activity Relationships--
The initial rates for the
ACP-catalyzed hydrolysis of six aryl phosphates differing in the
leaving group pKa values (phenyl phosphate, 4-cyanophenyl phosphate, 3-chlorophenyl phosphate, 4-nitrophenyl phosphate,
-naphthyl phosphate, and
L-phosphotyrosine) were determined. Measurements were
performed at pH 5.3 and 37 °C using the concentration range
0.05-3.2 mM for all substrates tested.
The Vmax and Km values
were obtained by solving the Michaelis-Menten equation
v = Vmax
[S]/(Km + [S]) by nonlinear regression analysis
with the aid of a computer program (Fig. P, BioSoft). We
examined the effect of the leaving group pKa
(pKa range, 7.14-10.07) on
kcat and
kcat/Km regarding the aryl
phosphatase activity of ACP (Brønsted correlation). These free energy
relationships provide information about the rate-limiting step of the
catalytic process and the nature of the transition state. Fig.
3 shows the Brønsted plots that
correlate, respectively, kcat and
kcat/Km values to the
pKa values of the substrate leaving group. The
left side of this figure shows the findings of earlier experiments on
kcat and
kcat/Km dependence on the
leaving group of seven acyl phosphates (24), whereas the right side
shows the results obtained here for kcat and
kcat/Km dependence on the
leaving group pKa of six aryl phosphates. For aryl phosphates, we found that the dependence of both log
kcat and log
kcat/Km on the leaving group
pKa are monophasic (Fig. 3, right).
By fitting the data to the linear equations
|
(Eq. 6)
|
or
|
(Eq. 7)
|
where pK is the pKa of the
leaving group XOH, we calculated, respectively, the
1g
values for kcat (-0.07 ± 0.02) and
kcat/Km (- 0.20 ± 0.11). The very small dependence of kcat and
kcat/Km on the leaving group
pKa for aryl phosphates contrasts with the strong dependence of kcat and
kcat/Km on the leaving group
pKa for acyl phosphates (
1g =
1.38 ± 0.14, and
1g =
1.44 ± 0.22, respectively (see left side of Fig. 3, which shows data for
the strictly homologous bovine enzyme; taken from Paoli et al. (24)). For aryl phosphates, the small dependence of
kcat on the structures of leaving group, which
differ markedly in the pKa (7.14 for
4-nitrophenyl phosphate and 10.07 for phosphotyrosine) indicates that
the kcat values for various aryl phosphates are very close to each other, suggesting that a common intermediate complex
is formed and accumulated before the limiting step of the catalytic
process takes place. No such accumulation of intermediate complex can
be seen from the data relative to the hydrolysis of acyl phosphates
catalyzed by the bovine CT-ACP (24), which is about 90% identical in
sequence with the human CT acylphosphatase.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of leaving group
pKa on kcat
and
kcat/Km
regarding CT acylphosphatase. The lines were drawn using a linear
regression method. Data for aryl phosphates are reported on the
right; the experimental points refer to 4-nitrophenyl
phosphate (7.14), 4-cyanophenyl phosphate (7.95), 3-chlorophenyl
phosphate (9.08), -naphtyl phosphate (9.24), phenyl phosphate
(9.99), and L-phosphotyrosine (10.07). Data for acyl
phosphates are shown on the left. The experimental points
are taken from Paoli et al. (30) and refer to bovine CT-ACP,
which has about 90% sequence identity with respect to the human
enzyme. The pKa value for each leaving group
is indicated in parentheses.
|
|
His-25 Is Involved in the Phosphotransfer and the Inorganic
Phosphate-H218O Oxygen Exchange Reactions
Catalyzed by ACP--
Previous experiments have demonstrated that
human CT-ACP was inactivated by the Woodward reagent K, which reacts
specifically with His-25 located in the active site region, even though
the enzyme contains other histidine residues and also several acidic residues that are potential targets for this reagent (25). This finding
revealed a very reactive nucleophile in the active site region. We thus
performed partition experiments with the H25Q mutant of human CT-ACP in
the presence and absence of 1 M glycerol. Table I shows
that the mutation of His-25 to Gln eliminates the enzyme capacity to
catalyze the phosphotransfer reaction. Interestingly, as observed in
the wild-type ACP, 1 M glycerol enhances the activity of
the H25Q mutant (by ~40% at pH 5.3 and ~25% at pH 6.5),
suggesting that the presence of glycerol also causes structural
modifications to occur in this mutant. Furthermore, Fig. 2D
shows that the mutation of His-25 to Gln in ACP causes a strong
decrease in the catalytic rate of the inorganic
phosphate-H218O oxygen exchange reaction. Taken
together, all of these results suggest that His-25 is involved in the
formation of an enzyme phosphohistidine intermediate during the
catalytic hydrolysis of pNPP.
 |
DISCUSSION |
Our results show evidence that human CT acylphosphatase catalyzes
the hydrolysis of aryl phosphate monoesters by a mechanism that
involves a nucleophilic catalysis step. On the contrary, no
nucleophilic catalysis occurs in the enzymatic hydrolysis of acyl
phosphates. We obtained consistent proof of our conclusions. First, the
results obtained by kinetic experiments performed at both pH 5.3 and
6.5 in the presence and absence of glycerol suggest that an
E-P covalent intermediate is formed during the catalytic hydrolysis of pNPP but not of benzoyl phosphate (see Table I). These
findings also suggest that the hydrolysis of E-P is the limiting step of pNPP hydrolysis by CT-ACP. In fact, if the
rate-limiting step is the formation of E-P (Scheme 1) and
the acceptor reacts with the intermediate after the rate-limiting step,
it does not increase the overall rate of pNPP breakdown. Only if the
rate-limiting step is the hydrolysis of E-P, will the
addition of the acceptor nucleophile increase the breakdown of the
intermediate and hence enhance the overall reaction rate (14, 20).
Furthermore, the results of experiments performed at increasing
glycerol concentrations (range, 0.2-1 M) agree with the
model described in Scheme 1 and Equation 4; this model assumes
that an E-P covalent intermediate is formed in the catalytic
pathway of aryl phosphate hydrolysis. In the 0-0.2 M
glycerol concentration range, fluorescence spectroscopy shows that
glycerol interacts with the enzyme and enhances its activity (see Fig
1). We also demonstrate that the mutation of His-25 to Gln completely
eliminates the capacity of the enzyme to catalyze the
transphosphorylation from pNPP to glycerol (Table I) demonstrating that
His-25 is involved in the nucleophilic catalysis step.
The second proof derives from the results obtained by experiments
with inorganic phosphate-H218O oxygen
exchange. These also demonstrate that an E-P covalent intermediate is formed in the catalytic pathway (Fig. 2). Indeed, the
inorganic phosphate-H218O oxygen exchange can
occur only if an E-P covalent intermediate is formed and is
hydrolyzed by cycling the two terminal step in the overall catalytic
process shown in Scheme 2. The mutation of His-25 to Gln eliminates the
capacity of the enzyme to catalyze the inorganic
phosphate-H218O oxygen exchange (Fig.
2D), suggesting that a phosphohistidine is involved in the process.
The third proof comes from the Brønsted free energy relationship
experiments. For different aryl phosphates, the Brønsted plot of
kcat versus the leaving group
pKa shows effectively constant kcat values (Fig. 3, right),
indicating that a common intermediate is formed before the
rate-limiting step of the catalytic pathway (see Scheme 1). The slope
of the curve is slightly negative, giving a
1g value
close to zero (-0.07). This value indicates that a very low amount of
negative charge develops on the leaving group oxygen in the transition
state. A flat leaving group dependence suggests two hypotheses: either
the protonation of the leaving group occurs at the transition state by
general acid catalysis, or alternatively, the hydrolysis step that
leads to the release of phosphate is not rate-limiting. The latter
hypothesis is ruled out because CT-ACP is able to catalyze
transphosphorylation from pNPP to glycerol; only if the
E-P covalent complex (Scheme 1) accumulates before the
rate-limiting step can the addition of a nucleophile (glycerol)
competing with water increase the overall substrate breakdown (Table
I).
In contrast with the results on aryl phosphates, previous findings on
acyl phosphates (24) have demonstrated that the structure of the acyl
group can have a dramatic effect on the rate of CT-ACP catalysis
(
1g value of
1.38). The difference of 1.34 units of the leaving group pKa for acetyl phosphate
(4.75) and p-nitrobenzoyl phosphate (3.41) leads to a
50-fold enhancement of kcat for
p-nitrobenzoyl phosphate versus acetyl phosphate
(24). Considering both substrate types (acyl phosphates and aryl
phosphates), a break in the leaving group dependence (see Fig. 3) is
usually attributed to a change in either the mechanism or the
rate-limiting step. The results of our experiments support the former.
Indeed, the earlier model for the hydrolysis mechanism of acyl
phosphates by CT-ACP, based on x-ray crystallography data, does not
include any step of nucleophilic catalysis (12). The mechanism proposed
by Thunnissen et al. (12) also agrees with our results
because we observed no transphosphorylation from benzoyl phosphate to
glycerol, consistent with the absence of any E-P covalent
intermediate in the catalytic pathway of acyl phosphate hydrolysis.
Collectively, all of our findings suggest that CT-ACP-catalyzed
hydrolyses of the two kinds of substrates follow two different pathways
(as shown in Scheme 3).

View larger version (7K):
[in this window]
[in a new window]
|
Scheme 3.
Two different pathways for acyl phosphate
(top) and aryl phosphate (bottom)
hydrolysis by ACP. E, enzyme; Ac, acyl;
Ar, aryl; P, phosphate group.
|
|
For acyl phosphates, a nucleophilic water molecule attacks the
Michaelis complex directly, producing Pi and the
carboxylate anion. On the contrary, for aryl phosphates, the
nucleophilic His-25 attacks the phosphorus atom of the substrate in the
Michaelis complex, phosphorylating the enzyme. The hydrolysis of the
E-P covalent complex concludes the process and is
rate-limiting. The difference among the two mechanisms could depend on
the fact that the carboxylate anion is a better leaving group than the
phenolate anion. For acyl phosphates, this may favor the formation of a strongly dissociative transition state in the limiting step (as indicated by the strong dependence of kcat on
the leaving group pKa; see Fig. 3,
left), which is efficiently stabilized by the active site
environment. This conclusion is supported by previous findings obtained
for the strictly homologous bovine CT-ACP (about 90% sequence
identity), which show that the formation of the transition state during
benzoyl phosphate hydrolysis is accompanied by a strong reduction in
the entropy of the hydrated enzyme-transition state complex compared
with that of the hydrated enzyme-substrate Michaelis complex (24).
The binding of ACP to pNPP is not influenced by the H25Q mutation; at
pH 5.3 we found a Km = 1.03 ± 0.13 mM for the mutant and a Km = 1.10 ± 0.04 mM for the wild-type enzyme. In contrast, the
binding of ACP to benzoyl phosphate is significantly affected by the
His-25 mutation; at pH 5.3, we found Km = 0.55 ± 0.03 mM for the mutant and Km = 0.16 ± 0.01 mM for the wild-type enzyme. Furthermore,
the mutation H25Q causes a significant decrease in the specificity
constant kcat/Km for both
pNPP and benzoyl phosphate. These findings indicate that His-25 is
involved in the catalysis of both kinds of substrates, although the
results reported in this study suggest that this residue plays
different roles in the two different pathways. These and other
findings, such as the observation that aryl phosphates behave as
competitive inhibitors with respect to acyl phosphates (data not
shown), collectively demonstrate that the enzyme possesses a unique
active site. The unusual behavior of CT-ACP with regard to the
mechanism of hydrolysis of two different kinds of substrates could be
related to the strongly different chemical properties of the leaving
groups (carboxylate and phenolate), which favor the formation of
different transition states in the respective chemical steps of the
catalytic hydrolysis processes. Results from the Brønsted analysis of
acylphosphatase-catalyzed reactions (Fig. 3) clearly indicate a change
in the nature of the transition state with a change the nature of the
substrates. We think that for acyl phosphates, the intrinsic high
stability of the carboxylate anion leaving group in the transition
state is the driving force of the hydrolytic reaction, whereas for aryl
phosphate monoesters a proton must be donated to the leaving group in
the transition state for a productive forward reaction.
Previously, we demonstrated that Woodward's reagent K
(N-ethyl-5-phenylisoxazolium-3'-sulfonate) is able to form a
Michaelis-like complex with human CT-ACP and then inactivate the
enzyme modifying His-25 (25). Woodward's reagent K interacts with the
substrate binding site by its sulfonic group moiety, as suggested by
the fact that some competitive inhibitors elicit protective actions against inactivation. The chemical modification of His-25 by
Woodward's reagent K is relatively fast and highly specific, because
other residues in the ACP molecule were not modified (25). The fact that Woodward's reagent K, which behaves as a substrate-like compound, binds to the CT-ACP active site and reacts easily with His-25 agrees
with the presence of this strongly reactive nucleophilic residue in the
active site environment. In fact, His-25 is very close to Arg-23,
an essential residue involved in substrate binding (12).
The cellular function of acylphosphatase is still under debate. In our
opinion, the most interesting results on the physiological function of
the enzyme in metazoan organisms are those demonstrating its capability
of uncoupling the membrane ion pumps acting on the aspartyl phosphate
intermediate formed during their functions. Nevertheless, the enzyme
seems to be implicated in other processes such as differentiation and
apoptosis (4-6). Transient expression of ACP in cells induces
apoptosis (26), and thus it has been hitherto impossible to obtain
stable clones overexpressing the enzyme. An understanding of all of the
aspects of the action mechanism of acylphosphatase may help in the
design and construction of specific enzyme inhibitors useful for
revealing the true substrates of acylphosphatase in differing cell
types and thus toward discovering its function.
 |
ACKNOWLEDGEMENTS |
We thank Gloria Borgogni for technical
support. We are also indebted to Drs. Gloriano Moneti and Giuseppe
Pieraccini of the "Centro Interdipartimentale di Servizi di
Spettrometria di Massa," University of Florence, Italy.
 |
FOOTNOTES |
*
This work was funded in part by the Consiglio Nazionale
delle Ricerche (CNR Target Project on Biotechnology) and in part by the
Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR-CNR, Biotechnology Program L.95/95).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dipartimento di
Scienze Biochimiche, Università degli Studi di Firenze, Viale Morgagni 50, 50134 Firenze, Italy. Tel.: 39-055-413765; Fax:
39-055-4222725; E-mail: camici@scibio.unifi.it.
Published, JBC Papers in Press, October 29, 2002, DOI 10.1074/jbc.M206918200
 |
ABBREVIATIONS |
The abbreviations used are:
ACP, acylphosphatase;
CT, organ-common type isoenzyme;
pNPP, p-nitrophenyl phosphate;
pNP, p-nitrophenol;
E-P, enzyme-phosphate;
MS, mass spectrometry.
 |
REFERENCES |
1.
|
Nediani, C.,
Marchetti, E.,
Nassi, P.,
Liguri, G.,
and Ramponi, G.
(1991)
Biochem. Int.
24,
959-968[Medline]
[Order article via Infotrieve]
|
2.
|
Nassi, P.,
Nediani, C.,
Liguri, G.,
Taddei, N.,
and Ramponi, G.
(1991)
J. Biol. Chem.
266,
10867-10871[Abstract/Free Full Text]
|
3.
|
Cecchi, C.,
Liguri, G.,
Pieri, A.,
Degl'Innocenti, D.,
Nediani, C.,
Fiorillo, C.,
Nassi, P.,
and Ramponi, G.
(2000)
Mol. Cell. Biochem.
211,
95-102[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Berti, A.,
Degl'Innocenti, D.,
Stefani, M.,
and Ramponi, G.
(1992)
Arch. Biochem. Biophys.
294,
261-264[Medline]
[Order article via Infotrieve]
|
5.
|
Chiarugi, P.,
Raugei, G.,
Marzocchini, R.,
Fiaschi, T.,
Ciccarelli, C.,
Berti, A.,
and Ramponi, G.
(1995)
Biochem. J.
311,
567-573[Medline]
[Order article via Infotrieve]
|
6.
|
Chiarugi, P.,
Degl'Innocenti, D.,
Taddei, L.,
Raugei, G.,
Berti, A.,
Rigacci, S.,
and Ramponi, G.
(1997)
Cell Death Differ.
4,
334-340[CrossRef]
|
7.
|
Raugei, G.,
Degl'Innocenti, D.,
Chiarugi, P.,
Solito, E.,
Modesti, A.,
and Ramponi, G.
(1999)
Biochem. Mol. Biol. Int.
47,
127-136[Medline]
[Order article via Infotrieve]
|
8.
|
Chiarugi, P.,
Degl'Innocenti, D.,
Raugei, G.,
Fiaschi, T.,
and Ramponi, G.
(1997)
Biochem. Biophys. Res. Commun.
231,
717-721[CrossRef][Medline]
[Order article via Infotrieve]
|
9.
|
Raugei, G.,
Modesti, A.,
Magherini, F.,
Marzocchini, R.,
Vecchi, M.,
and Ramponi, G.
(1996)
Biotechnol. Appl. Biochem.
23,
273-278[Medline]
[Order article via Infotrieve]
|
10.
|
Taddei, N.,
Stefani, M.,
Vecchi, M.,
Modesti, A.,
Raugei, G.,
Bucciantini, M.,
Magherini, F.,
and Ramponi, G.
(1994)
Biochim. Biophys. Acta
1208,
75-80[Medline]
[Order article via Infotrieve]
|
11.
|
Taddei, N.,
Stefani, M.,
Magherini, F.,
Chiti, F.,
Modesti, A.,
Raugei, G.,
and Ramponi, G.
(1996)
Biochemistry
35,
7077-7083[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Thunnissen, M. M. G. M.,
Taddei, N.,
Liguri, G.,
Ramponi, G.,
and Nordlund, P.
(1997)
Structure
5,
69-79[Medline]
[Order article via Infotrieve]
|
13.
|
Camici, G.,
Manao, G.,
Cappugi, G.,
and Ramponi, G.
(1976)
Experientia
32,
535[Medline]
[Order article via Infotrieve]
|
14.
|
Zhang, Z. Y.,
and Van Etten, R. L.
(1991)
J. Biol. Chem.
266,
1516-1525[Abstract/Free Full Text]
|
15.
|
Fiaschi, T.,
Raugei, G.,
Marzocchini, R.,
Chiarugi, P.,
Cirri, P.,
and Ramponi, G.
(1995)
FEBS Lett.
367,
145-148[CrossRef][Medline]
[Order article via Infotrieve]
|
16.
|
Deng, W. P.,
and Nickoloff, J. A.
(1992)
Anal. Biochem.
200,
81-88[Medline]
[Order article via Infotrieve]
|
17.
|
Sanger, F.,
Nicklen, S.,
and Coulson, A. R.
(1977)
Proc. Natl. Acad. Sci. U. S. A.
74,
5463-5467[Abstract]
|
18.
|
Ramponi, G.,
Treves, C.,
and Guerritore, A.
(1966)
Experientia
22,
705-706
|
19.
|
Baginski, E. S.,
Foa, P. P.,
and Zak, B.
(1967)
Clin. Chim. Acta
15,
155-158[CrossRef]
|
20.
|
Fersht, A.
(ed)
(1999)
Structure and Mechanism in Protein Sciences
, pp. 216-244, W. H. Freeman and Co., New York
|
21.
|
Zhao, Y., Wu, L.,
Noh, S. J.,
Guan, K. L.,
and Zhang, Z. Y.
(1998)
J. Biol. Chem.
273,
5484-5492[Abstract/Free Full Text]
|
22.
|
Zhang, Z. Y.,
Malachowski, W. P.,
Van Etten, R. L.,
and Dixon, J. E.
(1994)
J. Biol. Chem.
269,
8140-8145[Abstract/Free Full Text]
|
23.
|
Vincent, J. B.,
Crowder, M. W.,
and Averill, B. A.
(1992)
Trends Biochem. Sci.
17,
105-110[CrossRef][Medline]
[Order article via Infotrieve]
|
24.
|
Paoli, P.,
Cirri, P.,
Camici, L.,
Manao, G.,
Cappugi, G.,
Moneti, G.,
Pieraccini, G.,
Camici, G.,
and Ramponi, G.
(1997)
Biochem. J.
327,
177-184[Medline]
[Order article via Infotrieve]
|
25.
|
Paoli, P.,
Fiaschi, T.,
Cirri, P.,
Camici, G.,
Manao, G.,
Cappugi, G.,
Raugei, G.,
Moneti, G.,
and Ramponi, G.
(1997)
Biochem. J.
328,
855-861[Medline]
[Order article via Infotrieve]
|
26.
|
Giannoni, E.,
Cirri, P.,
Paoli, P.,
Fiaschi, T.,
Camici, G.,
Manao, G.,
Raugei, G.,
and Ramponi, G.
(2000)
Mol. Cell. Biol. Res. Commun.
3,
264-270[CrossRef][Medline]
[Order article via Infotrieve]
|
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.