Protein-tyrosine phosphatases (PTPases) catalysis
involves a cysteinyl phosphate intermediate, in which the phosphoryl
group cannot be transferred to nucleophiles other than water. The dual specificity phosphatases and the low molecular weight phosphatases utilize the same chemical mechanism for catalysis and contain the same
(H/V)C(X)5R(S/T) signature motif present in
PTPases. Interestingly, the latter two groups of phosphatases do
catalyze phosphoryl transfers to alcohols in addition to water. Unique to the PTPase family are two invariant Gln residues which are located
at the active site. Mutations at Gln-446 (and to a much smaller extent
Gln-450) to Ala, Asn, or Met (but not Glu) residues disrupt a
bifurcated hydrogen bond between the side chain of Gln-446 and the
nucleophilic water and confer phosphotransferase activity to the
Yersinia PTPase. Thus, the conserved Gln-446 residue is responsible for maintaining PTPases' strict hydrolytic activity and
for preventing the PTPases from acting as kinases to phosphorylate undesirable substrates. This explains why phosphoryl transfer from the phosphoenzyme intermediate in PTPases can only occur to water and not to other nucleophilic acceptors. Detailed kinetic analyses also suggest roles for Gln-446 and Gln-450 in PTPase catalysis. Although Gln-446 is not essential for the phosphoenzyme formation step, it plays an important role during the hydrolysis of the
intermediate by sequestering and positioning the nucleophilic water in
the active site for an in-line attack on the phosphorus atom of the
cysteinyl phosphate intermediate. Gln-450 interacts through a bound
water molecule with the phosphoryl moiety and may play a role for the
precise alignment of active site residues, which are important for
substrate binding and transition state stabilization for both of the
chemical steps.
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INTRODUCTION |
Protein-tyrosine phosphatases
(PTPase)1 consist of a family
of enzymes that catalyze the removal of phosphoryl groups on tyrosine residues in proteins that are introduced by protein- tyrosine kinases.
So far, approximately 100 PTPases have been identified, and the
predicted total number of human PTPases may reach 500 based on the
available genome sequencing data (1, 2). Amino acid sequence alignment
of PTPases from bacteria, yeast, and mammalian organisms indicates that
the only structural element that has amino acid sequence identity among
all PTPases corresponds to the catalytic domain that spans over 250 residues (3). The hallmark feature that defines the PTPases is the
active site sequence (H/V)C(X)5R(S/T)
in the catalytic domain also known as the PTPase signature motif (4).
Work from a number of laboratories has led to the conclusion that
PTPase catalysis proceeds through a double displacement mechanism
involving a covalent phosphoenzyme intermediate. The side chain of the
active site Cys residue serves as a nucleophile to accept the
phosphoryl group from the phosphotyrosine in a substrate and form a
kinetically competent cysteinyl phosphate intermediate (5, 6). The
active site Arg residue interacts with the phosphoryl moiety of the
substrate and plays a role in both substrate binding and transition
state stabilization (4, 7). In addition, the conserved hydroxyl group
of the Ser/Thr in the PTPase signature motif is essential for efficient
hydrolysis of the phosphoenzyme intermediate (8, 9). To facilitate substrate turnover, PTPases also employ an invariant Asp residue acting
as a general acid/base catalyst (3, 9).
Interestingly, the catalytic strategy employed by PTPases is also
shared by the dual specificity phosphatases and the low molecular
weight phosphatases, which share little sequence identity with the
PTPases (10, 11). The only similarities among these three groups of
phosphatases are the relative placements of the essential cysteine,
arginine, and serine/threonine residues in the active sites that
constitute the PTPase signature motif
(H/V)C(X)5R(S/T). Despite
the lack of sequence identity, the spatial arrangements of the
catalytic Cys, Arg, Ser/Thr, and Asp residues are conserved (12, 13),
consistent with the common catalytic mechanism. However, it is also
apparent that even though these phosphatases utilize the same mechanism
to catalyze phosphate monoester hydrolysis, they differ in active site
specificity and exhibit measurably different structure of the
transition state (14). To understand the structural and biochemical
origins for these functional differences, we have begun to investigate
the role of amino acids that are conserved only in a specific
phosphatase subfamily.
The pathogenic bacteria Yersinia encodes a PTPase essential
for its virulence (15). Because of its extraordinary phosphatase activity, the Yersinia PTPase has been the archetype of the
PTPase family for mechanistic investigations. Site-directed mutagenesis of conserved residues in the Yersinia PTPases combined with
detailed kinetic analyses have provided important insight into residues that are essential for PTPase catalysis (3, 4, 8). In this paper, we
examined the functional role of two invariant glutamines (Gln-446 and
Gln-450 in the Yersinia PTPase) located at the C terminus of
the PTPase catalytic domains (Fig. 1). As
shown in the crystal structures, the two Gln residues are situated at
the active site (16, 17). There are no corresponding Gln residues in
the dual specificity phosphatases and the low molecular weight phosphatases. The crystal structures of the Yersinia PTPase
complexed with oxyanion such as tungstate or sulfate have shown that
the apical anion oxygen (O1) is coordinated by the side chain of
Gln-446 (Fig. 2 and Refs. 18 and 19).
Both Gln-446 and Gln-450 form hydrogen bonds with a conserved
structural water, WAT1 (WAT505 in PTP1B, Ref. 20), which also interacts
with the apical oxygen and one of the phosphoryl oxygens (O2) in the
oxyanion (Fig. 2). The apical oxygen in the oxyanion occupies the
position that is equivalent to the phenolic oxygen of the leaving group
in the phosphoenzyme formation step or the oxygen atom of the attacking nucleophilic water molecule in the phosphoenzyme hydrolysis step. Furthermore, the structure of the catalytic inactive C215S mutant PTP1B
with phosphotyrosine bound indicates that the side chain of Gln-262
(equivalent to Gln-446 of the Yersinia PTPase) forms part of
the phenyl ring binding pocket, and the N
2 of Gln-266 interacts with
WAT505, which makes a hydrogen bond with the phenolic oxygen of
phosphotyrosine (20). In this paper, we demonstrate the functional
importance of the two invariant Gln residues in PTPase-catalyzed
reaction.

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Fig. 1.
Sequence alignment of the PTPase catalytic
domains starting from the active site signature motif to the C terminus
including the invariant glutamine residues. The sequences shown
here were retrieved from the GenBankTM using GCG program:
Yop51 (the Yersinia PTPase) (387-468, accession no.
M30457), hPTP1B (207-297, accession no. M31724), mSHPTP (445-529,
accession no. M90389), PTP-MEG (844-926, accession no. M68941),
PTP-PEST (223-313, accession no. M93425), PTP_alpha (434-521,
accession no. M34668), PTP_gamma (217-304, accession no.
X54132), PTP_epsilon (327-414, accession no. X54134), rLAR
(1496-1583, accession no. L11587), and CD45 (682-769, accession no.
Y00062). The conserved residues are shown in bold. The
invariant glutamine residues are marked with arrows using
the numbers from the Yersinia PTPase.
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Fig. 2.
Active site features of the
Yersinia PTPase detailing the interactions of Gln-446 and
Gln-450. The numbers along the dashed lines are
distances (in Å) taken from the crystal structure of the
Yersinia PTPase complexed with tungstate (Brookhaven Protein Data Bank accession code 1YTW).
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EXPERIMENTAL PROCEDURES |
Materials--
p-Nitrophenyl phosphate
(pNPP) was obtained from Fluka. Ethylene glycol,
-naphthyl phosphate, 4-methyl umbelliferyl phosphate, and phenyl
phosphate were purchased from Sigma. Aryl phosphate monoesters,
4-acetylphenyl, 4-cyanophenyl, 4-ethoxycarbonylphenyl, 4-fluorophenyl,
4-bromophenyl, 4-chlorophenyl, 4-methylphenyl, 4-ethyl phosphate, and
4-methoxylphenyl phosphate, were synthesized as described (21).
Solutions were prepared using deionized and distilled water. Deuterium
oxide was from Aldrich. Site-directed mutagenesis kit was from Bio-Rad,
and DNA sequencing kit from U. S. Biochemical Corp.
Site-directed Mutagenesis--
Substitutions at residues Gln-446
and Gln-450 of the Yersinia PTPase were made by
site-directed mutagenesis with the procedure of Kunkel et
al. (22). The oligonucleotide primers used were as follows: Q446A,
GGTATTATGGTAGCAAAAGATGAGC; Q446E,
TATTATGGTAGAAAAAGATGA; Q446 M,
GGTATTATGGTAATGAAAGATGAGCAA; Q446N,
GGTATTATGGTAAATAAAGATGAGCAA; Q450A,
CAAAAAGATGAGGCACTTGATGTTC; Q450E,
AAAAGATGAGGAACTTGATGT; Q450 M,
CAAAAAGATGAGATGCTTGATGTT CTG. All of the mutations were verified by DNA sequencing.
Expression and Purification of the Recombinant
Phosphatases--
The wild type Yersinia PTPase and the
mutants Q446A, Q446E, Q446M, Q446N, Q450A, Q450E, Q450M were expressed
under the control of T7 promoter in Escherichia coli BL21
(DE3) grown at 23-25 °C after induction with 0.4 mM
isopropyl-1-thio-
-D-galactopyranoside, and recombinant
proteins were purified to near homogeneity as described (23). The
recombinant dual specificity phosphatase VHR was expressed in E. coli and purified to homogeneity as described previously (24). The
yeast low molecular weight phosphatase Stp1 was expressed in E. coli and purified to homogeneity as described previously (25).
Urea Denaturation--
The urea-induced Yersinia
PTPase denaturation was studied by monitoring the decrease in the
intrinsic tryptophan fluorescence at 340 nm (slit width, 5 nm) with an
excitation wavelength of 295 nm (slit width, 3.5 nm). The experimental
details were described in Zhang et al. (23).
Steady-state Kinetics--
Initial rates for the hydrolysis of
pNPP and other aryl phosphate monoesters by the
Yersinia PTPases was measured as described previously (8).
Buffers used were as follow: pH 3.8-5.7, 100 mM acetate;
pH 5.8-6.5, 50 mM succinate; pH 6.6-7.3, 50 mM 3,3-dimethylglutarate; and pH 7.5-9.0, 100 mM Tris. All of the buffer systems contained 1 mM EDTA and the ionic strength of the solutions were kept
at 0.15 M using NaCl. The enzyme active site concentration
was treated as the protein concentration determined from the absorption
at 280 nm using the coefficient
A2801 mg/ml of 0.352 (8). The
Michaelis-Menten kinetic parameters were determined from a direct fit
of the v versus [S] data to the Michaelis-Menten equation
using the nonlinear regression program KinetAsyst (IntelliKinetics, State College, PA). Inhibition constants for the Yersinia
PTPases by arsenate were determined as described previously (4).
Pre-steady-state Kinetics--
Pre-steady-state kinetic
measurements of the Gln-446 and Gln-450 mutant Yersinia
PTPases catalyzed hydrolysis of pNPP were conducted at pH
6.0 and 4.5 °C. The reaction was monitored by the increase in
absorbance at 410 nm of the p-nitrophenolate product. The
enzyme concentrations were 42 µM for Q446A and 49 µM for Q450A, and the pNPP concentration was
20 mM. The details for data collection and analysis were as
described (8).
Detection of Reaction Products by 31P
NMR--
Dephosphorylation of pNPP in the presence of
phophoryl acceptor (1 M ethylene glycol) was monitored on a
Varian VXR 500 MHz spectrometer operating at 202.3 MHz using the
following parameters: acquisition time, 3.0 s; pulse width, 37 ms;
delay time, 1.0 s; spectral width, 8,000 Hz. All spectra were
recorded with proton broad band decoupler on. The spectrometer was
locked on D2O resonance line. The buffer used contained 50 mM succinate (pH 6.0), 1 mM EDTA, 1 M ethylene glycol, and 20% D2O. The chemical
shift of inorganic phosphate in the same buffer was set to zero. The
concentration of pNPP was 20 mM, and the
reaction was initiated with the addition of a catalytic amount of
enzyme (1.5 - 2.0 × 10
7 M) in 3 ml of
the buffer mentioned above. NMR data were collected at two time points
of the reaction (at which the sample was incubated for 5 min in boiling
water to inactivate the enzyme): one at 1 min to 1.5 h (wild type
enzyme at 1 min, Q446A at 30 min, Q450A at 1.5 h, etc.), the other
at 12 h. Multiple scans were taken to reduce the noise.
Protein Phosphatase Assays--
The phosphorylated substrate
(myelin basic protein (MBP), GST-Elk, Kemptide, or GST-Rb), diluted in
phosphatase buffer (20 mM HEPES, pH 7.0, 1 mM
EDTA, 1 mM dithiothreitol), was added to an equal volume of
phosphatase (wild type or mutant Yersinia PTPase), also in
phosphatase buffer, to a final volume of 20 µL. This phosphatase reaction mixture was incubated at 37 °C for one hour. For MBP, GST-Elk, and GST-Rb, 20 µL of SDS sample buffer was added to each reaction mixture to quench the phosphatase reaction. Half of this final
mixture was resolved on 15% SDS gel and subjected to autoradiography. For Kemptide, all of the reaction mixture at the end of 1 h of incubation was blotted onto P81 filter paper (Whatmann). The filters were then washed four times with 80 mM phosphoric acid for
10 min each, washed with 95% ethanol, dried, and counted in the
scintillation counter.
The amount of wild type or mutant Yersinia PTPase used was
normalized to an equal amount of phosphatase activity in each of the
reactions. The following amounts of each phosphatase was used for all
experiments except for the second round of Rb dephosphorylation, where
different amounts of the same phosphatases were incubated for two
different time points to determine concentration and time dependence of
possible dephosphorylation observed in the first phospho-Rb
dephosphorylation experiment. The amount of phosphatase used (in µg)
was 0.84, wild type; 1.08, Q446A; 0.64, Q446E; 3.35, Q446M; 3.08, Q446N; 5.72, Q450A; 57.0, Q450E; and 5.5, Q450M.
Preparation of Phosphorylated Protein Substrates--
1) MBP and
GST-Elk (a transcription factor phosphorylated by extracellular
signal-regulated kinase-1). GST-ERK1 (1.5 µg), GST-super
mitogen-activated protein or extracellular signal-regulated kinase (0.5 µg), 10 µCi of [
-32P]ATP and either 10 µg of MBP
or 10 µg GST-Elk were mixed together in the kinase buffer (18 mM HEPES, pH 7.4, 10 mM magnesium acetate, 50 µM ATP) to a final volume of 20 µL, and was incubated
at 30 °C for 1 h. The kinase reaction was quenched by the
addition of 80 µL of phosphatase buffer. Ten µL of this mixture was
used for phosphatase assay. 2) Kemptide (synthetic peptide as substrate of protein kinase A, Leu-Arg-Arg-Ala-Ser-Leu-Gly). Protein kinase A
(catalytic subunit) was mixed with 10 µg of the synthetic peptide, Kemptide, and 20 µCi of [
-32P]ATP in 50 µL of the
protein kinase A kinase buffer (20 mM Tris, 10 mM dithiothreitol, 5 mM NaF, 10 mM
MgOAc, 200 µM ATP). This mixture was incubated at
30 °C for 1 h to allow phosphorylation of the Kemptide. The
kinase reaction was quenched by the addition of 200 µL of the
phosphatase buffer. Ten µL of this mixture was then used for
phosphatase assay. 3) GST-Rb (C-terminal domain of retinoblastoma tumor
suppressor protein). GST-CDK4 and GST-cyclin D2 (1 µg each) were
activated by incubation in 40 µL of proliferating Jurkat cell lysate
(supplemented with 1 mM ATP) at room temperature for 1 h. The activated kinase was affinity purified on glutathione-agarose and eluted in 25 mM HEPES, pH 7.0, containing 10 mM glutathione. Half of the eluent (10 µL) was mixed with
10 µL of the cyclin-dependent kinase kinase buffer (50 mM HEPES, pH 7.0, 10 mM MgCl2, 5 mM MnCl2, 1 mM dithiothreitol, 20 µM ATP) containing 10 µg of GST-Rb and 10 µCi
[
-32P]ATP. This reaction mixture was incubated at
37 °C for 1 h. The kinase reaction was quenched by the addition
of 80 µL of the phosphatase buffer, and 10 µL of this mixture was
used for phosphatase assay.
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RESULTS |
Physicochemical Properties of the Gln-446 and Gln-450 Mutant
Yersinia PTPases--
To evaluate the role of the conserved Gln-446
and Gln-450 in PTPase catalysis, these Gln residues were altered by
site-directed mutagenesis. The carboxyamide side chain does not ionize
at physiological pHs but is relatively polar, being capable of both
donating and accepting hydrogen bonds. The Gln residue was changed to
(a) Glu, which is isosteric with Gln but can ionize as
the pH is raised from the acidic range, (b) Asn, which is
the lower homolog (one methylene group shortened) with the carboxyamide
group retained, (c) Met, which is similar in size to Gln but
lacks the carboxyamide group, and (d) Ala, which completely
eliminates the Gln side chain. All of the mutations were verified by
DNA sequencing. Initially, the mutant Yersinia PTPases were
expressed in E. coli as described for the wild type enzyme,
i.e. 6 h growth at 37 °C after 0.4 mM isopropyl-1-thio-
-D-galactopyranoside induction (23).
Like the wild type, Q446E remained soluble in the cell lysate. However, more than half of the total recombinant Q446A, Q446N, and Q450E protein
was found in the pellet, and the majority of the expressed protein for
mutant Q446M, Q450M, and Q450A became insoluble at the same expression
conditions. Subsequently, when the cells were grown at room temperature
overnight after
isopropyl-1-thio-
-D-galactopyranoside induction, the
amount of the soluble protein obtained increased markedly. All of the
mutant PTPases were purified to near homogeneity as judged by
SDS-polyacrylamide gel electrophoresis, using procedures identical to
those described for the wild type enzyme (23).
The Gln-446 and Gln-450 mutants had chromatographic and ultraviolet
absorption spectral characteristics similar to those of the wild type.
The
max values of the fluorescence emission spectra of
the mutants were the same as the wild type. To assess the
conformational stability of the mutants, urea-induced denaturation was
studied (see "Experimental Procedures"). As can be seen in Table
I, the free energies of unfolding for
Q446E and Q450E are similar to the wild type, whereas they are
significantly reduced for the Q446A and Q450A mutants. Collectively,
the side chains of Gln-446 and Gln-450 may be involved in the
appropriate folding process and contribute to the overall stability of
the enzyme.
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Table I
Unfolding free energy changes of the Yersinia PTPases by urea
All measurements were done in 50 mM succinate, 1 mM EDTA, [I] = 0.15 M buffer at pH 6.0 and
25°C. Parameters characterizing the urea denaturation were determined
by fitting the data to G°obs = G°N U + mG[Urea] (36), where
G°N U is the free energy change for protein
unfolding at zero urea concentration, [Urea]1/2 is the
concentration of urea at which half of the protein is denatured. Errors
were standard errors derived from direct fitting of the data to the
equation.
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Steady-state Kinetic Properties of the Yersinia PTPases--
Table
II summarizes the steady-state kinetic
parameters for the wild type and the mutant enzymes at 30 °C and
several pH values using p-nitrophenyl phosphate
(pNPP) as a substrate. The competitive inhibition constants
for the binding of arsenate to the Yersinia PTPases are also
listed in Table II. There are several points that are worth mentioning.
First, although Glu is isosteric with Gln, the Q450E mutant exhibited
kcat and
kcat/Km values that were
30-60-fold and 100-1000-fold lower than those of the wild type enzyme
at every pH examined. Furthermore, the binding affinity of Q450E for
arsenate decreased by 30-fold as the pH was raised from 5 to 7. Interestingly, substitution of Gln-446 by a Glu residue had minimal
effect on the kcat values, although both the
Km for pNPP and the Ki
for arsenate increased by severalfold as the pH was raised. Second,
with the exception of Q450E, substitutions at Gln-446 and Gln-450
generally had rather modest effects on the kinetic parameters, although the effects were greater for the Gln-450 mutants than the Gln-446 mutants. For example, substitutions at Gln-446 generally led to less
than 10- and 5-fold reduction in kcat and
kcat/Km values, respectively.
In contrast, substitutions at Gln-450 resulted in a decrease in
kcat by 10-fold and
kcat/Km by 10-100-fold. Third, with the exception of Q446E and Q450E, the affinity of the
native and mutant PTPases toward arsenate did not change appreciably with pH, and alterations at positions 446 and 450 did not lead to
significant changes in the Ki values.
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Table II
Kinetic parameters of the Yersinia PTPase and its conserved Gln mutants
Errors for kcat, Km, and
Ki values were standard errors calculated by
nonlinear least squares fits of the data to appropriate
Michaelis-Menten equations. For each experiment, eight substrate
concentrations covering the range of 0.2-5 Km were
used.
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Leaving Group Dependence--
The leaving group dependence of the
wild type Yersinia PTPase as well as the Q446A and Q450A
mutants was investigated using a series of aryl phosphates. To minimize
nonspecific steric effects, only para-substituted phenyl
phosphates were used. Fig. 3 shows the
Brønsted plots which relate the logarithms of
kcat and
kcat/Km, respectively, to the
pKa values of the leaving groups for
4-nitrophenyl, 4-acetylphenyl, 4-cyanophenyl,
4-ethoxycarbonylphenyl, 4-fluorophenyl, 4-bromophenyl, 4-chlorophenyl,
4-methylphenyl, 4-ethyl phosphate, and 4-methoxylphenyl phosphate.
Linear least-squares fitting of the Brønsted plots yielded the
slopes, which correspond to the
lg values. The
lg values describe the leaving group dependence of the
reaction and can often be regarded as an approximate measure of the
extent of bond cleavage between the reaction center and the leaving
group. For the wild type enzyme, the
lg for
kcat was 0.036 ± 0.032, whereas the
lg for kcat/Km
was
0.19 ± 0.051. Similarly, Q446A exhibited a
lg for kcat of
0.011 ± 0.011, and a
lg for
kcat/Km of
0.12 ± 0.050, whereas Q450A exhibited a
lg for
kcat of
0.013 ± 0.037, and a
lg for kcat/Km
of
0.13 ± 0.053. Thus, elimination of the carboxyamide functional group from residues 446 or 450 had negligible effect on the
values.

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Fig. 3.
Effect of aryl phosphate leaving group
pKa on kcat (A)
and kcat/Km (B)
for the wild type ( ), Q446A ( ), and Q450A ( )
Yersinia PTPase. The lines were drawn by a linear regression method (Kleidagraph, Abelbeck Software).
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Pre-steady-state Analysis--
The overall mechanism of hydrolysis
of aryl phosphates catalyzed by the Yersinia PTPase involves
a number of steps that are represented schematically in Scheme
1.
The reaction proceeds through a
sequence involving binding of substrate which is then cleaved with
phosphoryl transfer (k2) to the nucleophilic Cys
residue. Subsequent base-catalyzed reaction with water cleaves the
phosphoenzyme intermediate (E-P) (k3) and release of phosphate completes the catalytic cycle. Burst kinetics was
observed with the Yersinia PTPase at pH 6.0 and 3.5 °C
using pNPP as a substrate (8). It was concluded that under
these conditions the rate-limiting step corresponds to E-P hydrolysis. Burst kinetics was also observed at pH 6.0 and 4.5 °C with all of
the Gln mutants characterized. The stopped flow traces for the Q446A
and Q450A mutant reactions are shown in Fig.
4. The observed rate constant for E-P
formation (k2) in the Q446A and Q450A reactions
was slightly higher (1.5-2-fold), whereas the rate constant for E-P
hydrolysis (k3) was lowered by 4-5-fold in
comparison with those of the wild type reaction reported in a previous
study (8). It is not clear whether the slightly higher burst rate is
significant since rate constants in the range of several hundreds per
second are barely within the detection limit of a typical stopped flow
spectrophotometer. Collectively, these results suggest that alteration
of either Gln-446 or Gln-450 does not change the rate-limiting step of
the dephosphorylation of pNPP by the enzyme.

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Fig. 4.
Burst kinetics observed with Q446A and Q450A
using pNPP as a substrate at pH 6 and 4.5 °C. The
pNPP concentration was 20 mM. Each stopped flow
trace was an average of six to eight individual experiments. The
specific rate constants were analyzed by fitting the experimental data
directly to the theoretical equation (the solid line):
[p-nitrophenolate] = At + B(1 e bt) + C through the use of the
nonlinear least squares algorithm in KISS (Kinetic Instruments, Inc.).
For Q446A, k3 and k4 were 538 s 1 and 13.5 s 1, respectively, and for
Q450A, k3 and k4 were 721 s 1 and 10.1 s 1, respectively.
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Substitutions at Gln-446 Confer Phosphotransferase Activity to the
Yersinia PTPase--
PTPase catalysis involves a cysteinyl phosphate
intermediate that can only be hydrolyzed by water. Alcohols, such as
methanol, ethanol, ethylene glycol, and glycerol, are not phosphoryl
acceptors in the PTPase-catalyzed reaction. For example, when the
Yersinia PTPase was incubated with pNPP in the
presence of 1 M ethylene glycol at pH 6.0, no phosphoryl
group transfer to the alcohol was observed and only the hydrolysis
product, inorganic phosphate, was detected using 31P NMR
(Fig. 5A). In contrast, when
the same experiment was performed with the Q446A, Q446N, or Q446M
mutant, in addition to hydrolysis, ethylene glycol phosphate was
generated during the process of dephosphorylation of pNPP
(data for Q446A is shown in Fig. 5B). Phosphoryl transfer to
alcohol also occurred with the Q450A and Q450M mutants, although only a
minute amount of ethylene glycol phosphate could be detected even after
prolonged incubation with the mutant phosphatases (data for Q450A is
shown in Fig. 5C). Interestingly, like the wild type enzyme,
neither Q446E nor Q450E was able to produce the alkyl phosphate product
(data not shown).

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Fig. 5.
Phosphoryl transfer reaction catalyzed by the
Yersinia PTPase monitored by 31P NMR
spectroscopy. Panels A, B, and C are
the NMR spectra of reactions catalyzed by the native, Q446A, and Q450A
Yersinia PTPase, respectively. The chemical shift of
inorganic phosphate is set to zero. The chemical shift of
pNPP is 1.4 ppm and that of ethylene glycol phosphate is
1.3 ppm.
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Dual Specificity Phosphatases and Low Molecular Weight Phosphatases
Possess Phosphoryl Transfer Activity--
Dual specificity
phosphatases and low molecular weight phosphatases also proceed through
a phosphocysteine intermediate and utilize a strategy similar to that
of the PTPases for phosphate monoester hydrolysis. Interestingly,
31P NMR analysis of the dual specificity phosphatase VHR
and the low molecular weight phosphatase Stp1 catalyzed pNPP
hydrolysis in the presence of alcohol clearly demonstrate transfer of
the phosphoryl group onto alcohols (data not shown). The
structural basis for this additional phosphotransferase activity may be
suggested by the fact that the bound oxyanion in VHR (26) or the bovine low molecular weight phosphatase (27, 28) is not shielded from
bulk solvent as in the Yersinia PTPase (18) due to the absence of the corresponding Gln residues. It is likely that alcohols and water have equal access to the cysteinyl phosphate intermediate in
VHR and the low molecular weight phosphatases.
Partition Experiments--
The effect of ethylene glycol on the
kinetics and product distribution of the Yersinia
PTPase-catalyzed hydrolysis of pNPP was examined to
ascertain the rate-limiting step and to provide additional evidence for
phosphoryl transfer. In these experiments (Scheme
2),
the overall rate of substrate
turnover (hydrolysis plus phosphate transfer) was measured by the
production of p-nitrophenol whereas the hydrolysis rate was
measured by the production of inorganic phosphate (21). As shown in
Table III, the kinetic parameters of
pNPP hydrolysis determined by the p-nitrophenol assay were similar to those determined by the phosphate assay in the
absence of ethylene glycol. The kcat values for
the hydrolysis of pNPP as determined by the phosphate assay
in the absence of ethylene glycol were also close to those determined
in the presence of 1 M ethylene glycol, because the
molarity of water (~55.5 M) was not changed significantly
by the introduction of 1 M ethylene glycol.
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Table III
Effect of ethylene glycol
All measurements were performed at pH 6.0 and 30°C. The reported
errors were the standard deviation of the mean. For each experiment,
eight substrate concentrations covering the range of 0.2-5
Km were used.
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The overall rate of the substrate turnover
(kcat) as measured by the production of
p-nitrophenol is given by Equation 1. When the rate-limiting
step is the hydrolysis of E-P, Equation 1 can be reduced to Equation 2.
Thus, the presence of ethylene glycol will increase the rate of
E-P breakdown and thus accelerate the overall reaction rate (Scheme 2 and Equation 2), provided ethylene glycol can serve as a phosphoryl
acceptor.
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(Eq. 1)
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(Eq. 2)
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It turned out that the presence of ethylene glycol did not have
any effect on the rate of catalysis by the wild type and the mutants
Q446E and Q450E, since they were unable to catalyze the transfer of a
phosphoryl group to an alcohol. However, as shown in Table III, mutant
PTPases that could catalyze phosphoryl transfer to alcohols as judged
by the 31P NMR data did exhibit increased overall reaction
rate in the presence of 1 M ethylene glycol. This was
especially so for the Q446A mutant for which the
kcat for overall reaction was accelerated 1.5-fold in the presence of 1 M ethylene glycol, consistent
with the relative amount of products generated as measured by
31P NMR (Fig. 5B). These results support that
E-P decomposition is the rate-limiting step for the hydrolysis of
pNPP catalyzed by Q446A, Q446N and Q446M.
To assess the significance of Gln-446 in controlling the nucleophile
specificity (H2O versus alcohol), we have
compared the rates of phosphotransfer to ethylene glycol for Q446A, the
dual specificity phosphatase VHR, and the low molecular weight
phosphatase Stp1. The second-order rate constant
(k4) for phosphotransfer can be determined from
Equation 2 by measuring the dependence of kcat
on the alcohol concentration (29). As shown in Fig. 6, k4 for Q446A is
21.6 M
1 s
1 which is 3 times
faster than that for Stp1 (6.6 M
1
s
1) and 45 times faster than that for VHR (0.48 M
1 s
1). Since the
Yersinia PTPase, VHR, and Stp1 exhibit different intrinsic
catalytic activities, we also compare the ratio of the rate of
phosphotransfer (k4) to the rate of hydrolysis
(k3' = k3/[H2O], Scheme 2 and
Ref. 29). The ratio
(k4/k3') is 0 for the
Yersinia PTPase, 19.4 for the Q446A mutant, 122 for Stp1 and 4.4 for VHR, indicating that the tendency to catalyze phosphoryl transfer to ethylene glycol is zero for the Yersinia PTPase,
moderate for VHR, and great for Stp1. Furthermore, replacement of
Gln-446 with an Ala has rendered the Yersinia PTPase with
considerable phosphotransferase potential
(k4/k3') that is 4.4-fold
higher than VHR and 6.3-fold lower than Stp1.

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Fig. 6.
Dependence of the overall rate of turnover
(kcat) on the concentration of ethylene glycol
at pH 6.0 and 30 °C. A, the Yersinia PTPase
Q446A; B, Stp1; and C, VHR.
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Do Mutations at the Two Gln Residues Alter the Yersinia PTPase
Substrate Specificity?--
The Yersinia PTPase displays
strict specificity toward Tyr(P)-containing peptides/proteins (30).
This specificity for Tyr(P) has been proposed to be due to the depth of
the amphipathic Tyr(P) binding pocket that exactly matches the length
of Tyr(P) (20). Since the side chain of Gln-262 (equivalent to Gln-446
of the Yersinia PTPase) is positioned at the rim of the
pocket and interacts with the aromatic ring of Tyr(P) (Ref. 20 and Fig.
7A), elimination of the side
chain may open the binding pocket such that phosphorylated Ser/Thr-containing peptides/proteins may also be able to serve as
substrates. A number of Ser-phosphorylated proteins (MBP, GST-Elk, Kemptide, or GST-Rb) were prepared and tested as potential substrates for the wild type as well as the Gln-446 and Gln-450 mutants (see "Experimental Procedures"). No significant dephosphorylation was observed for any of the substrates tested (data not shown).

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Fig. 7.
A proposed model for the two chemical steps
in the PTPase-catalyzed reaction. A, phosphoenzyme
formation; and B, phosphoenzyme hydrolysis. The drawings are
based on the published structural data on the PTPase-substrate
complexes and PTPase-oxyanion complexes (17-20), the energy-minimized
atomic models of the noncovalent enzyme-substrate complex, the
phosphoenzyme intermediate (19), and the kinetic data presented in this
paper.
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DISCUSSION |
The function of the two conserved glutamines in the active site of
PTPases has been probed by site-directed mutagenesis. These two Gln
residues are unique to the PTPase family and no corresponding residues
are present in the dual specificity phosphatases and the low molecular
weight phosphatases. In the structure of PTP1B/C215S bound with
substrate, the side chain of Gln-262 (equivalent to Gln-446 in
Yersinia PTPase) interacts with the phenyl ring of the
phosphotyrosine (Tyr(P)), and defines a portion of the rim for the
Tyr(P) binding pocket (20). A similar interaction between Gln-446 and
Tyr(P) is observed in an energy minimized model of the
Yersinia PTPase with Tyr(P) bound (19). In the structures of
PTPases bound with substrates or oxyanions, a trapped structural water
molecule, WAT1 in the Yersinia PTPase and WAT505 in PTP1B, is observed (19, 20). WAT1 forms hydrogen bonds with the side chains of
the invariant residues Gln-446 and Gln-450, O-1 and O-2 of the
oxyanion, and the carboxylate of the general acid/base Asp-356 in the
PTPase-oxyanion complexes (Fig. 2). In the PTPase-substrate complexes,
this water molecule makes hydrogen bonds with the side chain of
Gln-450, the tyrosine leaving group oxygen (the scissile oxygen), O-2
of the phosphate, and the carboxylate of the general acid/base Asp-356
(Fig. 7A, and Ref. 20). Interestingly, a new water molecule,
WAT3, is identified in the Yersinia PTPase-nitrate complex
as well as in an energy minimized model of the phosphocysteine intermediate (19). Nitrate is coplanar and lacks the apical oxygen
present in tetrahedral oxyanion such as tungstate. Instead, WAT3 sits
directly above the nitrate and is perfectly in line with the sulfur
atom of the active site Cys residue. WAT3 is proposed to be the
nucleophilic attacking molecule for hydrolysis of the cysteinyl
phosphate intermediate and is coordinated primarily by the side chains
of Gln-446, Gln-357, Asp-356, and WAT1 through hydrogen bonds (Fig.
7B, Ref. 19).
Based on the reported structural data, Gln-446 and Gln-450 likely
participate in both of the chemical steps, i.e.,
phosphoenzyme formation and decay. Gln-446 may function to help to
align the phenyl ring of Tyr(P) in the first step and to position the
attacking water in the second step. Gln-450 makes a hydrogen bond with
WAT1, which in turn is in hydrogen bonding distance with the apical oxygen (O-1) and one of the phosphoryl oxygen (O-2). Thus, Gln-450 may
play a role in proper stabilization of the metaphosphate-like transition state (31) as well as in leaving group stabilization and
nucleophile activation. For the Yersinia PTPase, the kinetic parameter kcat/Km is
primarily limited by the phosphoenzyme intermediate formation step
(31), whereas the kcat term is mostly determined
by intermediate breakdown. Results from kinetic analysis of the
site-directed mutants of Gln-446 and Gln-450 confirm some of the
structural data and provide additional insights into the mechanisms by
which these two residues effect catalysis.
Replacements of Gln-446 by Ala, Asn, or Met have minimal effect on
kcat/Km and only less than
10-fold reduction in kcat. In the case of
PTP1B/Q262A, kcat/Km and
kcat are reduced by 7- and 83-fold,
respectively, using the tyrosine-phosphorylated lysozyme as a substrate
(7). It is not clear why a larger reduction in
kcat is observed for the PTP1B/Q262A mutant. It
appears that Gln-446 does not play a significant role in the
phosphoenzyme formation step. As shown in Fig. 7B, the
putative nucleophilic water molecule is coordinated by a bidentate
hydrogen bond with the side chain of Gln-446. Such an interaction does
not exist in the Q446A, Q446N, and Q446M mutants so that the
positioning of the attacking water could be affected leading to a
reduced rate for the phosphoenzyme hydrolysis step. Indeed, within the PTP1B/Q262A phosphoenzyme intermediate, the position of the
nucleophilic water is displaced by 1.5 Å from being colinear with the
active site Cys-215 S
-P
bond.2 The moderate decrease
in rate for phosphoenzyme hydrolysis observed in the
Yersinia Gln-446 mutants is consistent with a dissociative transition state for which minimal activation of the nucleophilic water
is required (29). In addition, a water molecule may take the place of
the Gln-446 side chain and partially rescue its function in the second
step. A new water is found at the position of the side chain of Gln-262
in the structure of PTP1B/Q262A phosphoenzyme complex.2
This may also explain the fact that Gln-446 mutants possess similar oxyanion binding affinity as the wild type.
Replacements of Gln-450 by Ala or Met have larger effects on both the
kcat/Km and
kcat, consistent with the notion that it
interacts through WAT1 with a phosphoryl oxygen, and the leaving group
oxygen in the first step and a phosphoryl oxygen, and the attacking
water in the second step. The results obtained with Q446E and Q450E are
rather interesting. Although glutamic acid and glutamine are isosteric,
the Gln to Glu substitution at residue 450 manifests the most
deleterious effects with up to 1200-fold decrease in
kcat/Km, and 66-fold decrease in kcat, 20-fold increase in
Km for pNPP and 30-fold increase in
Ki for arsenate. Furthermore, these deleterious effects increase as the pH is raised. These results strongly suggest that in the native Yersinia PTPase, the carboxyamide side
chain must donate a hydrogen bond to WAT1 through its amide group (Fig. 7). WAT1 may receive another hydrogen bond from Asp-356 or the backbone
amide from Gln-357, and donate one hydrogen bond to a phosphoryl oxygen
and another hydrogen bond to the leaving group oxygen or the attacking
water (Fig. 7). Thus, deprotonation of the carboxylic acid at residue
450 in Q450E will cause detrimental effects due to charge repulsion. In
contrast, it appears that Gln-446 can be largely replaced by a Glu
residue with little change in kcat and only
moderate increase in Km for pNPP and Ki for arsenate.
Gln-446 Is the Residue Escorting the Nucleophilic Water--
The
ability to catalyze phosphoryl transfer to nucleophilic acceptors in
addition to water is a common feature for phosphate ester hydrolases,
such as nonspecific alkaline and acid phosphatases, that involve a
covalent phosphoenzyme intermediate (32). The PTPase-catalyzed reaction
proceeds through a covalent phosphocysteine intermediate that is
subsequently hydrolyzed by water. However, careful investigations from
this laboratory have shown that the Yersinia PTPase and the
human PTP1B can only catalyze the transfer of the phosphoryl group from
the intermediate to water, yielding inorganic phosphate. Similar
observations have also been made with other PTPases. For example,
LAR-D1 PTPase does not show a discernible tendency to transfer the
phosphoryl group to ethylene glycol or glycerol, and the CD45 catalytic
fragment displayed no phosphotransferase activity to alcohols as
cosubstrates (33). Glycerol or propane-1,2-diols, at concentrations of
4-6 M, accelerated the kcat of the
full-length SHP-1 by 47-fold and of the PTPase domain by 8-fold.
However, 31P NMR spectroscopy indicates no formation of
glycerol phosphate during hydrolysis of pNPP by SHP-1 in
50% glycerol (34). The increase in rate is likely caused by a glycerol
induced conformational change which alleviates the autoinhibited
state.
Alcohols are better nucleophiles than water for accepting a phosphoryl
group (29). The fact that alcohols such as methanol, ethanol, ethylene
glycol and glycerol are much better nucleophilic acceptors of
phosphoryl group than water and yet they fail to serve as phosphate
acceptors in the PTPase reaction indicates that the phosphoryl group in
the intermediate is only accessible by water. As shown in the
PTPase-oxyanion complexes, the side chain of Gln-446 interacts with the
apical oxygen of the oxyanion, which effectively shields the oxyanion
from the aqueous environment (18). In the PTPase-nitrate complex or the
energy-minimized model of the phosphoenzyme intermediate (19) the side
chain of Gln-446 coordinates the attacking nucleophilic water via a bidentate hydrogen bond. Thus, nucleophiles larger than water such as
alcohols cannot gain access into the active site due to steric
hindrance. Alternatively, it is also possible that both protons of the
nucleophilic water are required for proper positioning for the
nucleophilic attack. For example, the nucleophilic water can donate one
H-bond to O
1 of Gln-446 and the other to the side chain of Asp-356,
which is consistent with the carboxylate functioning as a general base
in the phosphoenzyme hydrolysis step (21). The nucleophilic water can
also receive a hydrogen bond either from N
2 of Gln-446 or from WAT1
(Fig. 7). Such interactions would position the water in an optimal
position for nucleophilic attack on the phosphorus atom (Fig. 7). Since
alcohols are incapable of making the same interactions and they can
only donate one H-bond, they cannot serve as phosphoryl acceptors.
Substitutions at Gln-446 by an Ala, an Asn, or a Met abolishes the
specific bidentate hydrogen bonding interactions with the nucleophilic
water and results in either smaller side chains (Q446A and Q446N) or a
side chain with an increased mobility (Q446M), such that alcohols can
replace the nucleophilic water and gain access to the phosphoenzyme
intermediate. This notion is supported by the observations that Q446A,
Q446N, and Q446M all displayed phosphoryl transfer activities. Q450A
and Q450M also exhibited minor phosphotransfer activities. This may be
caused by the slight misalignment in Gln-446 due to the disruption of
the interactions between Gln-450 and WAT1 (Fig. 7). Collectively, these
results strongly suggest that Gln-446 functions to escort and orient
the attacking water molecule for the hydrolysis of the phosphoenzyme intermediate. The isosteric substitution of Gln-446 by a Glu
residue introduces minimal alterations in the kinetic parameters and
does not confer phosphotransfer activity to the Yersinia
PTPase, suggesting that the Glu residue at 446 is protonated at pH 6.0 so that a similar bidentate hydrogen bond can be made with the
nucleophilic water. Interestingly, although substitution of Gln-450 by
a Glu residue leads to dramatic decrease in both catalytic efficiency and substrate/inhibitor affinity, the Q450E mutant still maintains the
hydrolytic activity with no phosphotransferase activity. This implies
that the Gln-450 to Glu substitution does not introduce drastic
structural alterations into the enzyme active site, especially in the
context of Gln-446.
Conclusions--
Regulation of enzyme activity by reversible
cycles of phosphorylation and dephosphorylation is now considered to be
one of the most important means by which cells respond to physiological stimuli. To ensure the precision and fidelity of this regulation, it
may be desirable that the PTPase only possesses hydrolytic activity and
protects the highly reactive phosphoenzyme intermediate from unwanted
nucleophilic acceptors (such as Ser or Thr residues in a protein)
during its course of action. It is shown here that Gln-446 is
responsible for keeping this unwanted "kinase" activity in check.
The PTPases, the dual specificity phosphatases and the low molecular
weight phosphatases employ a similar chemical mechanism for
phosphate monoester hydrolysis. However, in contrast to the PTPases, which possess no phosphoryl transfer activity, both
the low molecular weight phosphatases (21, 29, 35) and the dual specificity phosphatase VHR (this study) are able to catalyze the
transfer of the phosphoryl group from the intermediate to alcohol
nucleophiles as well as to water. The biological implication for this
observed mechanistic difference is unclear. Results from kinetic
characterization of the Gln-446 and Gln-450 mutant PTPases combined with structural information on PTPases support the
conclusion that the two invariant Gln residues constitute an integral
part of the PTPase active site, serving different roles in the
catalytic reaction. Gln-450 plays a role for the precise alignment of
active site residues, which are important for substrate binding and
transition state stabilization, whereas Gln-446 is important for the
optimal positioning of the nucleophilic water molecule in the
phosphoenzyme hydrolysis step.
We thank Dr. David Barford for helpful
comments on the manuscript, and Dr. Terry Dowd for assistance in NMR
experiments.