Protein and Peptide Letters, Vol. 11, No. 2, pp.

149-155, 2004
Bentham Science Publishers Ltd.
0929-8665/04 $ 45.00 + .00

CALCINEURIN HYDROLYSIS OF PARA-NITROPHENYL

PHOSPHOROTHIOATE

Donna J. Spannaus-Martin and Bruce L. Martin*

Department of Laboratory Medicine and Pathology, University of Minnesota

420 Delaware Street, SE; Mayo Mail Code 609; Minneapolis, MN 55455
Telephone: 612-625-5988; E-mail: marti285@umn.edu

Abstract: para-Nitrophenyl phosphorothioate (pNPT) was hydrolyzed by calcineurin at initial rates

slightly, but comparable to rates for para-nitrophenyl phosphate (pNPP). Kinetic characterization yielded
higher estimates for both K m and V max compared to pNPP. Metal ion activation of phosphorothioate
hydrolysis was more promiscuous. Unlike the hydrolysis of with pNPP, Ca 2+, Mg 2+, and Ba 2+ activated
calcineurin as well as Mn2+.

Keywords: Calcineurin, Metal-activated enzyme, Substrate specificity, Substrate analog.

INTRODUCTION

Protein phosphorylation is arguably the most commonly identified and the most important post-
translational modification of proteins found in the cell and has been implicated in virtually every process
involved in cellular regulation [1,2]. A critical aspect of the field is definition of the effect of the
phosphorylation or dephosphorylation reaction on the function of the target protein. One approach used to
identify target functions is the thiophosphorylation of proteins can be accomplished using ATP- -S as the
substrate for protein kinases in place of ATP. Thiophosphorylation generates modified proteins considered
resistant to protein phosphatases and has been exploited in cellular and biochemical studies of the
functions of phosphoproteins [3-9]. Thiophosphorylated proteins have been shown to be inhibitors of
many protein serine and protein tyrosine phosphatases [3,5,7]. Thio analogs of substrates have also been
used in mechanistic investigations of enzymatic [10-20] and chemical [21-23] hydrolyses of phosphate
esters. An analog of the common substrate, para-nitrophenyl phosphate (pNPP), para-nitrophenyl
phosphorothioate (pNPT) has a sulfur atom in place of a non-bridge oxygen and has been studied in some
detail [23]. Solution hydrolysis of the thioate is more rapid than the native compound, whereas the

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enzymatic reaction has been found to be slower [10-12,24,25]. For a subset of these enzymes, a chiral form
of pNPT has been used to explore the stereochemistry of the hydrolysis reaction [11,24].
Much effort has been given to investigating the mechanism of action of the protein phosphatase,
calcineurin. Early studies demonstrated properties that were consistent with the absence of a phosphoryl-
enzyme intermediate [26,27]. Protein tyrosine phosphatases and the nonspecific phosphatases catalyze the
hydrolysis of phosphate ester bond by the formation of phosphoryl-enzyme intermediate [28-31]. In
contrast, purple acid phosphatases are postulated to catalyze the direct hydrolysis of substrate using metal
ion activated water. Direct hydrolysis was confirmed for one form of purple acid phosphatases using chiral
pNPT to characterize the stereochemical course of the reaction [25]. As expected, resolution of the crystal
structure of calcineurin yielded a structure showing the presence of metal ions at the putative active site
[32,33] and provided for a mechanism consistent with that of the purple acid phosphatases [34,35]. Other,
evolutionary related protein serine phosphatases have been shown to have metal ions bound in the active
site [36,37]. It is likely that all of these enzymes use a similar mechanism. In this work, pNPT was found to
be hydrolyzed by calcineurin. The enzymatic reaction was characterized and compared to the hydrolysis of
pNPP. The implications for the mechanism of calcineurin are discussed.

EXPERIMENTAL PROCEDURES
MATERIALS

Buffers, EGTA, and phenyl-Sepharose were purchased from Sigma Chemical. The substrate pNPP
(Sigma 104 substrate) was also purchased from Sigma. The substrate analog pNPT was obtained from Drs.
Irina Catrina and Alvan Hengge (Utah State University; Ref. 20). DE-52 cellulose was obtained from
Whatman. Other chemicals (metal salts, etc.) were obtained from Fisher. Water and all buffer solutions were
treated with Chelex-100 to remove contaminating metal ions.

METHODS
Bovine brain calcineurin was isolated from bovine brain to apparent homogeneity using a
modification of the method of Sharma et al. [38] using MOPS buffer instead of Tris for the preparation of all
solutions. The final step was chromatography on immobilized calmodulin with elution by EGTA.
Calmodulin was purified by the procedure of Sharma and Wang [39], but including chromatography on
phenyl-Sepharose with elution also by EGTA [40]. The recovered proteins were dialyzed against metal ion
free solutions and protein concentrations were determined by the method of Bradford [41]. Standard
activity measurements were done at 30C with pNPP or pNPT in 25 mM MOPS, pH 7.0, 5-20 g/ml
calmodulin, and 5-20 g/ml calcineurin with 1.0 mM Mn2+. When used, other metal ions were added to 1.0
mM, and Mg 2+ was also tested at 20.0 mM. Calmodulin was added to the same concentration as calcineurin
in units of mg/ml; the resulting molar concentration was approximately 5-fold the molar concentration of
calcineurin. The reactions were monitored continuously at 410 nm on a Molecular Devices Spectramax 250
microtiter plate reader. Data points were collected for up to 20 minutes and activity measurements were
corrected for activity in the absence of added metal ion. Data reduction was done using the programs
Enzyme Kinetics (Trinity Software) and Deltagraph (SPSS, Inc.).
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RESULTS AND DISCUSSION

An analog of pNPP, para-nitrophenyl phosphorothioate was tested and found to be hydrolyzed by

calcineurin. In parallel assays, pNPT was hydrolyzed more rapidly than was pNPP as shown in Figure 1. That
the thioate compound could be hydrolyzed by calcineurin highlights differences between calcineurin and
other phosphatases. There is no thio effect observed for calcineurin. As shown in Figure 1, alkaline
phosphatase hydrolyzed pNPT at approximately 5% the velocity for pNPP. The lower activity for alkaline
phosphatase with pNPT has been characterized [19,56] with kinetic studies showing that kcat with pNPT is
70-100 times lower than measured with pNPP. Protein tyrosine phosphatases likewise show decreased
activity with pNPT compared to pNPP with the effects ranging up to a 5,000-fold difference in kcat values for
the Yersinia enzyme [18]. The decreased activity, designated the thio effect, has been ascribed to alterations
in the transition state for both enzymes. Recent studies have provided evidence that the interpretation may
not be so straightforward [19]. Calcineurin is unique among these enzymes in having higher activity with
pNPT, an apparent inverse thio effect.

Figure 1. Hydrolysis of pNPT by Calcineurin and Alkaline Phosphatase. The hydrolysis of pNPT (closed
bars) by calcineurin was assayed and compared to the hydrolysis of pNPP (open bars). Both substrates were
included at 20.0 mM with activity with pNPP set as 100%. Also shown are data for the hydrolysis of these
substrates by alkaline phosphatase. Control values (100%) for the hydrolysis of pNPP were 0.58 M pNP
released per minute with calcineurin as the catalyst and 49.8 M/min with alkaline phosphatase. Other
details are in the text.

Optimal calcineurin activity requires the inclusion of exogenous metal ion as an activator [42-48],
with Mn 2+ typically supporting the highest level of activity with pNPP. The characterization of the metal
ion activation of calcineurin has been an ongoing area of study [49-55] prompting a survey of metal
152

ions in supporting pNPT hydrolysis. Metal ions were found to activate hydrolysis of pNPT and with the
profile showed a more promiscuous response to the panel of metal ions tested. The profile of metal ion
activation of pNPT hydrolysis is shown in Table I as well as data for pNPP hydrolysis for a subset of metal
ions. With the exception of Zn 2+, all the metal ions supported higher activity with pNPT than with pNPP
when compared to Mn2+. Most notably, activation by the alkali earth metal ions showed the same level of
activity as that supported by Mn 2+. This is very different from activity measured with pNPP as Mg 2+ has
generally not supported activity very well compared to Mn 2+ when present at similar concentrations.
Activation by Mg2+ typically requires higher concentrations (20.0 mM) to support activity with pNPP. With
pNPT, however, increasing the concentration of Mg2+ from 1.0 to 20.0 caused a decrease in hydrolysis. Mg2+
was found to cause a decrease in the aqueous hydrolysis of pNPT [23]. This observed lack of selectivity for
metal ion activation likely results from the enhanced reactivity of the phosphorothioate and from a lack of
selectivity in the interaction of substrate with metal ion. Catrina and Hengge [23] established that both the
monoanion and dianion forms of pNPT were more readily hydrolyzed than the corresponding forms of
pNPP. These authors showed that metal ions (Mg2+ and Cd 2+) did not complex with pNPT. Mechanistically,
the thermodynamics of pNPT hydrolysis are likely easier to overcome. Previous studies established that
Mg 2+ activation of calcineurin had the greatest energy barrier compared to Mn2+ and other transition metal
ions.

Table I. Activation of pNPT and pNPP Hydrolysis by Exogenous Metal Ions.

pNPT Hydrolysis Percent pNPP Hydrolysis Percent

Metal ion Concentration mM Activity Activity

Ba2+ 1.0 94 2 not tested

Mg2+ 1.0 122 8 not tested
Mg2+ 20.0 64 2 32 2
Ca2+ 1.0 78 4 <1
2+
Sr 1.0 96 2 <1
Mn2+ 1.0 100 1 100.0
Co2+ 1.0 101 6 71 8
Ni2+ 1.0 86 5 72 1
2+
Cu 1.0 73 5 precipitate
Zn2+ 1.0 134 7 161 6
Activity was measured at 30C with pNPT (10.0 mM) as the substrate and corrected for non-enzymatic hydrolysis. All metal
ions were added as the chloride salts. Percent activity was standardized to the activity measured for each substrate with 1.0
mM Mn2+ set to100%. Shown is the standard error of the measurement. Data for calcineurin-catalyzed hydrolysis of pNPP
were taken from references 51 and 55.

In parallel with pNPP, the hydrolysis of pNPT was investigated using various concentrations to
characterize the kinetic parameters of the hydrolysis. Each substrate was varied over the range from 5.0 to
50.0 mM. Estimates for the kinetic parameters are given in Table II. Unfortunately, saturation was not
completely achieved with pNPT; increasing the concentration further was not possible because of
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solubility difficulties. As expected from initial assays, the maximal velocity was higher with pNPT. The
estimated value of Km was greatly elevated and is, therefore, very much an approximate value. The calculated
parameters do enable the calculation that the hydrolysis rate for 20 mM pNPT would be 18% higher than for
20 mM pNPP. This difference is consistent with the data shown in Figure 1.
As described, most phosphatases do not hydrolyze pNPT very well compared to pNPP as attributed
in some cases to poor transition state complementarity with the sulfur substrate. These data, however, have
been reported for phosphatases that exploit a mechanism different from calcineurin. Calcineurin and related
protein phosphatases, PP-1 and PP-2A, and the purple acid phosphatases do not catalyze the formation of an
enzyme-phosphoryl intermediate and use metal ions seemingly in the direct hydrolysis of the phosphate
ester. Protein tyrosine phosphatases and non-specific phosphatases do form a phosphoryl-enzyme
intermediate by exploiting a nucleophilic amino acid. In many cases, it is hydrolysis of this intermediate
that defines the net rate of the reaction. The substrate has minimal influence on kcat . Calcineurin catalysis is
effectively defined by the actual cleavage of the phosphate ester linkage and kcat is influenced by the nature
of the leaving [57]. The k cat for calcineurin catalyzed hydrolysis of these small substrates is much lower
than catalysis by protein tyrosine phosphatases or alkaline phosphatases.

Table II. Kinetic Characterization of pNPT and pNPP Hydrolysis.

Substrate Vmax (M mol/min) Apparent Km (mM) Predicted Rate @ 20 mM

pNPT 3.2 0.9 79 24 0.65 mM mol/min

pNPP 1.1 0.2 20 5 0.55 mM mol/min
Activity was measured at 30C with either pNPP or pNPT (varied) as the substrate and with 1.0 mM Mn2+ as the metal ion.
Measured rates were corrected for non-enzymatic hydrolysis. Parameters were estimated using the program Enzyme
Kinetics from Trinity Software.

Calcineurin has the ability to hydrolyze the ester linkage despite the presence of a sulfur in a non-
bridge position. There was an apparent inverse thio effect suggesting that there was enhanced interaction
with the phosphorothioate in the transition state compared to the phosphate substrate. Alternatively, the
enhanced turnover of the phosphorothioate may simply be because the sulfur substitution provides a better
leaving group, an important determinant for calcineurin catalysis [57]. Recognition of the substrate was
seemingly modulated more greatly inasmuch as the Km appears to be considerably greater than the typical
Km for pNPP having all oxygens. This was suggestive that the larger sized sulfur atom weakened
recognition of the substrate in the ground state. Activation by exogenous metal ions showed greater
promiscuity that was also likely because of the weaker interactions of pNPT with enzyme and metal ions. A
corollary conclusion is that exploiting protein thiophosphorylation in cells may not be suitable for
characterizing the targets or effects of calcineurin-catalyzed dephosphorylation. With the hydrolysis of
pNPT by calcineurin now established, what is now possible is the direct elucidation of the stereochemistry
of the calcineurin-catalyzed using chiral pNPT.
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ACKNOWLEDGMENTS

The authors are grateful to Drs. Irina Catrina and Alvan Hengge (Utah State University) for
providing the para-nitrophenyl phosphorothioate used in these experiments. This work was initiated while
the authors were affiliated with the University of Tennessee Health Science Center in Memphis, TN, and was
partially supported by funds from the American Heart Association (Award 9950840V to BLM).

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Received on August 25, 2003, accepted on December 19, 2003.