Diesterase activity and substrate binding in purple acid phosphatases

Article abstract of DOI:10.1021/ja072647q

The purple acid phosphatases (PAPs) are bimetallophosphatases that are reported to lack phosphodiesterase activity based on the absence of reactivity with bis(p-nitrophenyl) phosphate. Yet, model systems designed to mimic the PAP active site preferentiall

Full text of DOI:10.1021/ja072647q

Published on Web 07/17/2007
Diesterase Activity and Substrate Binding in Purple Acid Phosphatases
Robynn S. Cox, Gerhard Schenk,*,§ Nata sˇ a Miti c´ , Lawrence R. Gahan, and Alvan C. Hengge*
†
§
§
,†
Department of Chemistry and Biochemistry, Utah State UniVersity, Logan, Utah 84322-0300, and School of
Molecular and Microbial Sciences, UniVersity of Queensland, Brisbane 4072, Australia
Received April 16, 2007; E-mail: hengge@cc.usu.edu
Purple acid phosphatases (PAPs) are dinuclear monoesterases1,2
that are structurally diverse but with a conserved set of ligands to
the Fe3 -M metal centers (M ) Fe, Zn, or Mn). The identity
+
2+
2+
3
of the nucleophile and the substrate binding mode have been matters
1
,3-5
of controversy (Figure 1).
In one proposal, substrate binds to
the divalent metal and is attacked by a terminal Fe3 -bound
hydroxide. In an alternative mechanism, substrate coordinates first
to the divalent metal and then forms a bridging complex, followed
by nucleophilic attack by the µ-hydroxide. We report that the PAPs
from pig and kidney bean catalyze the hydrolysis of diesters if the
second ester group is small, and the kinetics of the reaction with
methyl p-nitrophenylphosphate suggest that a terminal-bound
hydroxide is the nucleophile for the diester, followed by attack of
the bridging hydroxide upon the resulting monoester without release
into solution.
+
Figure 1. Schematic diagram of two potential binding modes of a phosphate
ester to the metal center of PAPs. In A, the nucleophile is the Fe(III)-
coordinated hydroxide; in B, it is a bridging µ-hydroxide.
Table 1. Kinetic Data for PAP Hydrolysis of Methyl p-Nitrophenyl
Phosphate (MpNPP), Methyl Phosphate (MP), and p-Nitrophenyl
Phosphate (pNPP) by Pig PAP (red) and kbPAP (blue) (Standard
Errors are Shown in Parentheses)
The PAPs are reported to lack the ability to hydrolyze diesters,
based on the lack of activity with bis(p-nitrophenyl)phosphate
6
(
BpNPP). In contrast, model complexes designed to mimic the
PAP metal center hydrolyze diesters, while monoesterase activity
7
has not been reported. This suggests that the inability of PAP to
hydrolyze BpNPP might result from steric considerations. Thus,
we tested the PAPs from pig (also termed uteroferrin) and from
red kidney bean using two diesters bearing smaller second ester
groups, methyl pNPP (MpNPP) and ethyl pNPP (EpNPP). While
BpNPP gave negligible turnover, consistent with a previous report,6
the other two diesters, particularly MpNPP, are substrates although
Table 2. Inhibition Data
m
binding is poor. The pH dependence for kcat and kcat/K of the
reactions with MpNPP was followed by monitoring release of
p-nitrophenol (see Supporting Information, Figure S1). The pH
optima for monoesterase and diesterase reactions were the same;
data in Table 1 were obtained at the optimal pH of 4.9 for pig
PAP and pH 6.25 for kbPAP (see Supporting Information for
EpNPP and BpNPP data).
and kbPAPs respectively, with surprisingly high K
1). MP is a weak competitive inhibitor of the PAP-catalyzed
hydrolysis of pNPP and of MpNPP, with a K
of ∼100 mM (Table
2). These K values confirm the unexpectedly weak binding of MP.
As noted above, previously characterized substrates with low
millimolar K values are aryl and benzyl esters. It may be that the
PAP active site region recognizes the aromatic moiety in these
substrates. Studies of PAP inhibitors have shown that aromatic
groups are important in achieving tight binding.8
In the MpNPP reactions, the rates of formation of p-nitrophenol
and of inorganic phosphate decreased significantly after only a few
percent of the diester was consumed (see Figure 2 and Supporting
Information). Addition of pNPP immediately resulted in rapid
production of p-nitrophenol, showing that this was not due to loss
of enzymatic activity (see Supporting Information for details). The
m
values (Table
i
i
At its pH optimum, the kcat for MpNPP hydrolysis by pig PAP
is close to that for the monoester pNPP,5 while for kbPAP, the
MpNPP kcat is about half that for pNPP.
,8
m
The high diester K
m
values presumably result from steric
values
requirements of the second ester group.14 For comparison, K
m
of aryl and benzyl monoesters range from 1.25 to 5.8 mM.5
Inorganic phosphate is a competitive inhibitor of the reactions of
both PAPs with MpNPP, with K values close to the literature values
i
for the reactions with pNPP (Table 2).
The reaction of MpNPP is assumed to first yield p-nitrophenol
and methyl phosphate, on the basis of the large difference in the
a
pK values of the two potential leaving groups and the observation
i m
low K for inorganic phosphate and high K of the diester leads to
that PAP catalysis shows a significant dependence on the leaving
group (âlg ) -0.4 and -0.6 for pig and kbPAP, respectively).5
Methyl phosphate (MP), the presumed product of the diester
the expectation of significant product inhibition. This was confirmed
by an experiment in which enzyme was preincubated with an
amount of inorganic phosphate corresponding to that produced after
the first 5% of reaction. Addition of MpNPP substrate gave rise to
the same slow rate observed after ∼5% had been turned over in
the experiments without preincubation with phosphate.
-
1
reaction, is a substrate, with kcat values of 17 and 20 s for the pig
†
Utah State University.
University of Queensland.
§
9550
9
J. AM. CHEM. SOC. 2007, 129, 9550-9551
10.1021/ja072647q CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
substrate occurs (Figure 1A), with subsequent hydrolysis of the
first ester bond by the terminal Fe3 -bound hydroxide. This forms
MP at the active site poised for immediate and subsequent attack
by the µ-hydroxide (Figure 1B). When free MP is the substrate,
turnover may be partially limited by the displacement of hydroxide
from Fe3 to form the bidentate complex.
+
+
While the requirement for a very small second ester group for
diesterase activity means that this activity has no biological
relevance, the results reveal that the metal centers in pig and kbPAP
have similar inherent catalytic competence in both monoesterase
and diesterase hydrolysis, a capability that has not been reported
in model systems.
Figure 2. The rates of formation of inorganic phosphate (9) and of
p-nitrophenol (() from a reaction of pig PAP initiated with 15 mM MpNPP
in 0.1 M acetate buffer at pH 4.90 at 22 °C. Enzyme concentration was 13
nM, in a reaction volume of 500 µL. Similar results were obtained with
kbPAP (see Supporting Information).
Acknowledgment. This work was supported by a grant to A.H.
from the NIH (GM47297). G.S. and L.G. wish to thank the
Australian Research Council for funding (DP0558652). We thank
members of the Hengge lab for comments on the manuscript.
In the reactions of MpNPP, the production of inorganic phosphate
and of p-nitrophenol were followed to determine whether the two
reactions are processive (proceeding from a single binding event)
or whether methyl phosphate is released into solution prior to
Supporting Information Available: Experimental procedures,
kinetic methods, pH profiles, and kinetic data. This material is available
free of charge via the Internet at http://pubs.acs.org.
rebinding and hydrolysis. MpNPP and MP have similar a K
m
, and
MP has a much lower kcat/K . Thus, if the MP product is released,
m
then its recapture and hydrolysis to inorganic phosphate should lag
behind the formation of p-nitrophenol in the initial stages of the
reaction when [MpNPP] . [MP]. In a reaction with an initial
concentration of 15 mM MpNPP, the rates of formation of
p-nitrophenol and of inorganic phosphate were indistinguishable
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(
9
The kinetic results pose an apparent conundrum: the consecutive
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(
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7
hydroxide. If the diester reacts in this manner in the PAP reactions,
as shown in A of Figure 1, then nucleophilic attack will result in
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bidentate fashion, attack by the bridging hydroxide would produce
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4
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+
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While the second possibility cannot be ruled out, we pro-
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data and with previous observations. Phosphodiesters bind and
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3341-3348.
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2+
(14) The higher K
m
might also result from a charge difference; the diester has
takes place by rapid binding to Fe followed by rate-determining
a charge of -1, while a monoester, at the pH optimum for the PAP
reaction, will be a mixture of monoanionic and dianionic forms. Whether
the monoanion or the dianion is the substrate has not been determined,
but it is possible that a monoester monoanion would be deprotonated upon
coordination to the metal center, resulting in superior binding as the
dianion, a possibility not open to a diester.
3+
bridging to Fe . The second step is significantly slower when the
3+
Fe ligand to be displaced is hydroxide rather than water (pK
.8).13
We propose that the PAP diesterase reactions occur by a
processive mechanism, whereby monodentate coordination of the
a
)
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