From the
Protein-tyrosine phosphatases (PTPases) are believed to exhibit
restricted specificity toward phosphotyrosine. I demonstrate here that
both the Yersinia PTPase and rat PTP1 can dephosphorylate
alkyl phosphates such as flavin mononucleotide, pyridoxal 5`-phosphate, D-glucose 6-phosphate, DL-
Protein-tyrosine phosphatases (PTPases)
Although considerable effort has been devoted to studying the
biological function of PTPases, a detailed understanding of their
substrate specificity is lacking. Such knowledge is crucial for the
design and development of novel PTPase inhibitors containing elaborate
functionality. Specific, tight-binding PTPase inhibitors have not been
reported. It is generally accepted that PTPases exhibit a strict
substrate specificity toward phosphotyrosine-containing
proteins/peptides(7, 8, 9) . Studies using
synthetic phosphopeptides have demonstrated that PTPases display amino
acid sequence sensitivity surrounding the
phosphotyrosine(10, 11, 12, 13, 14) .
Little is known about the substrate specificity inherent within the
PTPase active site, namely the molecular features that enable PTPases
to favor aryl over alkyl phosphates. This is, in large part, due to our
incomplete understanding of the scope and limitation of the active-site
substrate specificity of PTPases, namely the range of molecular
moieties that can be readily accommodated and processed by the
catalytic apparatus of this family of enzymes. I am intrigued with
identifying those structural features on the substrate molecule that
enable PTPases to discriminate between aromatic and aliphatic phosphate
monoesters. In this study, I compare the reactivity of different aryl
and alkyl phosphates with the Yersinia PTPase and rat PTP1. My
results demonstrate that PTPases can dephosphorylate a variety of alkyl
phosphates, including phosphoserine and phosphothreonine. These
observations provide exciting new opportunities for mechanistic
investigations as well as PTPase inhibitor design.
That PTPases catalyze the hydrolysis of alkyl phosphates
was further evidenced by an independent method that utilizes
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy I have shown that the rate-limiting step of the PTP1-catalyzed
hydrolysis of aryl phosphates corresponds to the decomposition of the
phosphoenzyme intermediate(22) . The fact that the k
The Yersinia PTPase and rat PTP1 both catalyze the exchange
reaction between
The results described in this paper should also be
useful in developing strategies for selective inhibition and
inactivation of specific PTPases. In spite of the potential value that
specific PTPase inhibitors may offer for the study of signal
transduction pathways and for therapeutic intervention, no such agents
have been reported. Deactivation of a particular PTPase could be
achieved by designing mechanism-based inhibitors (or suicide
inhibitors) (25, 26, 27) that are PTPase
substrates and that, upon enzymatic transformation, specifically modify
the PTPase. Barford et al.(28) have proposed, on the
basis of the three-dimensional structure of human PTP1B, that
PTPases' specificity for phosphotyrosine-containing peptides
probably results from the depth of the active-site cleft since the
smaller phosphoserine and phosphothreonine side chains would not reach
the phosphate-binding site. However, I would expect that an alkyl
phosphate moiety with an appropriate distance between the peptide
backbone and the phosphate should make a reasonable PTPase substrate.
For example, an alkyl phosphate with six CH
I thank Dr. Terry Dowd for assistance with the
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-glycerophosphate, O-phospho-L-serine, and O-phospho-L-threonine. The k
values for alkyl phosphates are orders of magnitude slower than
those for aryl phosphates such as p-nitrophenyl phosphate and O-phospho-L-tyrosine, reflecting the intrinsic lower
chemical reactivity of the alkyl phosphates. In addition, the k
values for the PTPase-catalyzed hydrolysis of
alkyl phosphates are similar to the k
values for
the PTPase-catalyzed
O exchange reaction between inorganic
phosphate and water. I conclude that the rate-limiting step for the
hydrolysis of alkyl phosphates has changed to the phosphorylation of
the PTPases, i.e. the formation of the phosphoenzyme
intermediate. The implications of the results described in this report
in terms of studying the PTPase catalytic mechanism and their potential
application in developing selective PTPase inactivators are discussed.
(
)are signaling molecules that act in concert with
protein-tyrosine kinases to regulate a variety of fundamental cellular
processes such as cell growth, mitogenesis, metabolism, gene
transcription, cell cycle control, and the immune
response(1, 2, 3) . PTPases constitute a growing
family of enzymes (now >40 members, excluding species homologues)
that rival protein-tyrosine kinases in terms of structural diversity
and complexity. Unlike tyrosine-specific and serine/threonine-specific
kinases, which share conserved sequences in their catalytic domains,
PTPases show no sequence similarity to serine/threonine phosphatases or
the broad specificity phosphatases such as acid or alkaline
phosphatases(4, 5) . Although many PTPases are proteins
of >400 amino acids, their catalytic domains are usually contained
within a span of 250 residues referred to as the PTPase domain. This
domain is the only structural element that has amino acid sequence
identity among all PTPases from bacteria to mammals(6) .
Materials
p-Nitrophenyl phosphate,
-naphthyl phosphate, O-phospho-L-tyrosine, O-phospho-L-serine, O-phospho-L-threonine, pyridoxal 5`-phosphate, FMN
(riboflavin 5`-phosphate), D-glucose 6-phosphate,
-D-glucose 1-phosphate, DL-
-glycerophosphate, and O-phosphorylethanolamine were purchased from Sigma. Deuterium
oxide (99.9%) was obtained from Aldrich. Solutions were prepared using
deionized and distilled water. Homogeneous recombinant Yersinia PTPase and the catalytic domain of rat PTP1 were purified as
described(15, 16) .
Steady-state Kinetics
Initial rates for the
enzyme-catalyzed hydrolysis of phosphate monoesters were measured at 30
°C by the production of inorganic phosphate using a colorimetric
method described earlier(17, 18) . Buffers used were as
follow: pH 5.0, 100 mM acetate; pH 6.0, 50 mM succinate; and pH 7.0, 50 mM 3,3-dimethyl glutarate. All
of the buffer systems contained 1 mM EDTA, and the ionic
strengths of the solutions were kept at 0.15 M using NaCl.
Michaelis-Menten kinetic parameters were determined from a direct fit
of the vversus [S] data to the
Michaelis-Menten equation using the nonlinear regression program GraFit
(Erithacus Software).
P NMR
P NMR
measurements were performed on a Varian VXR 500 spectrometer operating
at 202.3 MHz. A spectral width of 20,000 Hz and an acquisition time of
1.5 s were used. All spectra were recorded under proton broad band
decoupling conditions. The spectrometer was locked on the deuterium
oxide resonance line of 20% D
O in the buffer solution. The
buffer used was a solution of 50 mM succinate, 1 mM EDTA with an ionic strength of 0.15 M at pH 6.0. Chemical
shift values were determined relative to 20 mM inorganic
phosphate in the same buffer. The Yersinia PTPase-catalyzed
hydrolysis of D-glucose 6-phosphate was carried out at 23
°C and pH 6.0 in the buffer described above containing 20%
D
O. The enzymatic reaction was initiated by the addition of
30 µl of 9.75 mg/ml Yersinia PTPase to the reaction
mixture (3.0 ml) containing 20 mMD-glucose
6-phosphate in the same buffer.
PTPases Are Active against Alkyl
Phosphates
Inorganic phosphate was produced as measured by the
well established phosphomolybdate colorimetric method (17, 18) when alkyl phosphates such as pyridoxal
5`-phosphate, D-glucose 6-phosphate, and DL--glycerophosphate were incubated with homogeneous
recombinant Yersinia PTPase or the mammalian PTPase, rat PTP1.
The amount of inorganic phosphate generated was proportional to the
amount of PTPase present and the duration of the reaction. All enzyme
assays were performed at 30 °C in buffers with a constant ionic
strength of 0.15 M containing 1 mM EDTA. EDTA was
also present in all buffers used for the purification of the
recombinant PTPases from Escherichia coli. Thus, it is
unlikely that the hydrolysis activity is due to a contaminating sample
of E. coli alkaline phosphatase, which requires Mg
and Zn
for activity. The following observations
suggest that the hydrolytic activity against alkyl phosphates is an
intrinsic property of PTPases. First, the hydrolysis of 10 mMDL-
-glycerophosphate by Yersinia PTPase could be
completely blocked by 1 mM vanadate, a commonly used PTPase
inhibitor. Second, DL-
-glycerophosphate inhibited the Yersinia PTPase-catalyzed hydrolysis of p-nitrophenyl
phosphate competitively at pH 7.0 with a K
of 20.4 ± 5.6 mM. Finally, when the
catalytically inactive Cys-403
Ser mutant Yersinia PTPase (which was overexpressed and purified identically to the
wild-type enzyme) was incubated with alkyl phosphates, no hydrolysis
was observed. Cys-403 in the Yersinia PTPase is the
active-site nucleophile that is essential for catalysis(9) .
Thus, the PTPase-catalyzed hydrolysis of alkyl phosphates is most
likely effected by the same active site that dephosphorylates aryl
phosphates.
P NMR to follow the production of inorganic phosphate. Fig. 1shows the
P NMR spectra of 20 mMD-glucose 6-phosphate before (lowerspectrum) and after (upperspectrum)
the addition of Yersinia PTPase. The
P chemical
shift of the inorganic phosphate in the same buffer system has been set
to zero. The two major peaks with chemical shifts of 1.82 and 1.94 ppm
in the absence of the Yersinia PTPase correspond to the
-
and
-anomers of D-glucose 6-phosphate, respectively.
Three hours after the addition of Yersinia PTPase (stock in pH
5.7 buffer), a new peak with a chemical shift value of 0.17 ppm
appeared in the upper spectrum, which corresponds to inorganic
phosphate. The slight variation in chemical shift from zero is most
likely due to the perturbation in pH caused by the introduction of
enzyme solution and the subsequent production of inorganic phosphate.
Consistent with this, the chemical shift values of the
- and
-anomers of D-glucose 6-phosphate have now changed to
1.86 and 1.98 ppm, respectively.
Figure 1:
P NMR spectra of 20 mMD-glucose 6-phosphate in 3 ml of 20% D
O, 50
mM succinate, 1 mM EDTA with an ionic strength of
0.15 M at pH 6. Other experimental conditions are specified
under ``Experimental Procedures.'' Lowerspectrum, no PTPase present; upper spectrum, 3 h
after 30 µl of 9.75 mg/ml Yersinia PTPase was added. The
P chemical shift of the inorganic phosphate in the same
buffer system has been set to zero.
summarizes the
specific activities of the Yersinia PTPase- and rat
PTP1-catalyzed hydrolysis of both aryl and alkyl phosphates measured at
20 mM substrate. It is apparent that PTPases can bring about
hydrolysis of not only aryl phosphates, but also alkyl phosphates such
as pyridoxal 5`-phosphate, DL--glycerophosphate, O-phospho-L-serine, and O-phospho-L-threonine. As expected, PTPases are much
more effective catalysts for the hydrolysis of aryl phosphates. For
example, the Yersinia PTPase dephosphorylates aryl phosphates
such as p-nitrophenyl phosphate and tyrosine phosphate
500-5000-fold faster than alkyl phosphates of primary alcohols such as
pyridoxal 5`-phosphate, flavin mononucleotide, D-glucose
6-phosphate, and DL-
-glycerophosphate. Interestingly,
alkyl phosphates of secondary alcohols (such as
-D-glucose 1-phosphate and O-phospho-L-threonine) or primary alcohols with
-substituted charges and/or side chains (such as O-phosphorylethanolamine and O-phospho-L-serine) are even poorer substrates than
esters of primary alcohols. This likely arises from the sensitivity of
the phosphorylation reaction to steric hindrance. For the rat
PTP1-catalyzed hydrolysis, p-nitrophenyl phosphate and
tyrosine phosphate are 4000-13,000-fold better substrates than
esters of primary alcohols such as D-glucose 6-phosphate and DL-
-glycerophosphate. Again, esters of secondary alcohols
or of primary alcohols with
-substitutions are even worse
substrates. Interestingly, pyridoxal 5`-phosphate and flavin
mononucleotide, which are primary alkyl phosphates, exhibit activities
similar to those of sterically hindered phosphate esters. Thus, rat
PTP1 is more sensitive to the nature of the substrates and displays a
more stringent active-site specificity than the Yersinia PTPase. The reason forPTPases' apparent low activity toward
alkyl phosphates may be due to either their intrinsic lower k
values or higher K
values for the alkyl phosphates, or both. I therefore
determined the Michaelis-Menten kinetic parameters associated with each
of the alkyl phosphates.
Kinetic Parameters for Alkyl Phosphates
The
PTPase-catalyzed hydrolysis of alkyl phosphates follows
Michaelis-Menten kinetics. lists the kinetic parameters
for the hydrolysis of aryl and alkyl phosphates by the Yersinia PTPase and rat PTP1 at pH 6.0 and 30 °C. In general, PTPases
display much higher k values toward aryl
phosphates than alkyl phosphates. For example, the k
values for the Yersinia PTPase-catalyzed hydrolysis of
aryl phosphates are 200-1000-fold faster than those of alkyl
phosphates. Similarly, the rat PTP1-catalyzed hydrolysis of aryl
phosphates exhibits 600-8000-fold higher k
values than that of alkyl phosphates. In contrast, PTPases show
lower K
values toward aryl phosphates
than alkyl phosphates. It is interesting to note that alkyl phosphates
with an aromatic moiety attached have K
values closer to those of aryl phosphates, suggesting that
the aromatic moieties are important for PTPase binding. Due to the
extremely slow rate of hydrolysis, the k
and K
values for
-D-glucose
1-phosphate, O-phosphorylethanolamine, O-phospho-L-serine, and O-phospho-L-threonine could not be accurately
estimated for the Yersinia PTPase and PTP1. For the same
reason, I could not accurately determine the kinetic parameters for the
PTP1-catalyzed hydrolysis of pyridoxal 5`-phosphate and flavin
mononucleotide.
The Rate-limiting Step for Alkyl Phosphate
Hydrolysis
It is well established that the PTPase-catalyzed
reaction involves a phosphoenzyme
intermediate(19, 20, 21) . This suggests that
the PTPase-catalyzed hydrolytic reaction is composed of both the
formation and the breakdown of a phosphoenzyme intermediate (). values for the PTPase-catalyzed hydrolysis of
alkyl phosphates are 2-3 orders of magnitude slower than those of
aryl phosphates suggests that the rate-limiting step for the hydrolysis
of alkyl phosphates is different from that of aryl phosphates. If both
classes of substrates go through the same rate-limiting step, namely
the hydrolysis of the common covalent phosphoenzyme intermediate, I
would expect to observe similar k
values for
both aryl as well as alkyl phosphates. I compares the pH
dependences of the kinetic parameters for the Yersinia PTPase-catalyzed hydrolysis of p-nitrophenyl phosphate
with those of pyridoxal 5`-phosphate, flavin mononucleotide, D-glucose 6-phosphate, and DL-
-glycerophosphate.
The maximal hydrolysis activity of alkyl phosphates centers around pH
6, while the pH maximum for the hydrolysis of p-nitrophenyl
phosphate is
5(6) . This is also consistent with a change
in the rate-limiting step for the hydrolysis of alkyl phosphates.
O-labeled phosphate and solvent water
(22, 23), which represents a partial reverse reaction of phosphate
monoester hydrolysis, namely P
to E
P
to E-P (see ). The k
values for exchange at pH 6.0 are 0.77 and 0.014 s
for Yersinia PTPase and PTP1, respectively, while k
values for the hydrolysis of p-nitrophenyl phosphate at pH 6.0 are 345 and 63.5
s
, respectively. Since the exchange rate is orders
of magnitude slower than the rate of phosphate monoester hydrolysis,
which is rate-limited by the breakdown of the phosphoenzyme
intermediate, I believe that phosphorylation of the enzyme by inorganic
phosphate has become rate-limiting for the
O exchange
reaction. Strikingly, the PTPase-catalyzed hydrolysis of alkyl
phosphates displays k
values that are very
similar to those of
O exchange between inorganic phosphate
and water. Mechanistically, the PTPase-catalyzed
O
exchange between inorganic phosphate and water resembles the
PTPase-catalyzed hydrolysis of alkyl phosphates since the
pK
values of the conjugated acid of the
leaving group, alkoxide (RO
) for alkyl phosphate and
hydroxide (HO
) for inorganic phosphate, are both
15. The formation of the phosphoenzyme intermediate involves the
attack by the active-site cysteine on the phosphorus atom and the
release of the leaving group, which can be phenoxide, alkoxide, or
hydroxide. The repulsion of an alkoxide or a hydroxide would require
much greater assistance from the enzyme than a phenoxide, which has
pK
values typically <10. Collectively,
my results indicate that the rate-limiting step for the
PTPase-catalyzed hydrolysis of alkyl phosphates is the formation of the
phosphoenzyme intermediate. The fact that the PTPase-catalyzed
hydrolysis of alkyl phosphates is very sensitive to steric properties
of the substrates is also consistent with this conclusion.
Mechanistic Implications
My observation that
PTPases dephosphorylate alkyl phosphates with a rate-limiting step that
corresponds to the phosphorylation of the enzyme and that is different
for aryl phosphates opens a new field for investigation of PTPase
catalysis. I can now divide the two chemical events, i.e. phosphorylation and dephosphorylation, and study them separately,
using alkyl phosphates and aryl phosphates, respectively. In the case
of the low molecular weight phosphatases, the utilization of alkyl
phosphates has led to the demonstration of a solvent-derived proton
``in flight'' in the transition state of the phosphorylation
process (24). Furthermore, one can ascertain the specific roles of
active-site residues in each catalytic step using site-directed
mutagenesis and alternative substrates. For example, PTPase catalysis
has been shown to involve general acid/base catalysis(6) . Since
the hydrolysis of aryl phosphates and phosphotyrosine-containing
substrates is rate-limited by the decomposition of a common
phosphoenzyme intermediate, it is difficult to determine the
contribution to catalysis of the intermediate formation by the putative
general acid/base. This should be possible with alkyl phosphates as
substrates.
units would
approximate the length of a tyrosine side chain. Since high affinity
substrate binding requires the presence of both the phosphorylated
residue and its surrounding amino acids, I believe that the
incorporation of relatively simple functionalities into a specific,
optimal phosphopeptide template should result in potent and selective
inactivators of PTPases. The fact that alkyl phosphate compounds are
much slower substrates and are rate-limited by the phosphorylation of
the enzyme suggests that the specificity determinants that are built
into the peptide-based suicide inhibitor can be fully exploited by the
enzyme. Systematic investigations of the parameters that affect
substrate binding and catalysis should yield deeper understanding of
the substrate specificity of PTPases. Such knowledge will facilitate
the design and development of specific PTPase inhibitors, which may
serve as new tools for studying signal transduction. Further studies
are also required to understand the biological significance of the
intrinsic alkyl phosphatase activity of PTPases in cellular signaling.
Table: 175597932p4in
ND, not detectable.
Table: pH dependences of kinetic parameters for
Yersinia PTPase-catalyzed phosphate monoester hydrolysis
P NMR data collection. I also thank Drs. John Blanchard
and Fred Brewer for comments on the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.