©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Are Protein-tyrosine Phosphatases Specific for Phosphotyrosine? (*)

Zhong-Yin Zhang (§)

From the (1)Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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--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.


INTRODUCTION

Protein-tyrosine phosphatases (PTPases)()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) .

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.


EXPERIMENTAL PROCEDURES

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% DO 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% DO. 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.


RESULTS AND DISCUSSION

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.

That PTPases catalyze the hydrolysis of alkyl phosphates was further evidenced by an independent method that utilizes 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% DO, 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 Kvalues 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 ().

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 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.

The Yersinia PTPase and rat PTP1 both catalyze the exchange reaction between O-labeled phosphate and solvent water (22, 23), which represents a partial reverse reaction of phosphate monoester hydrolysis, namely P to EP 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.

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 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: ND

  
Table: pH dependences of kinetic parameters for Yersinia PTPase-catalyzed phosphate monoester hydrolysis



FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant DRTC 5P60 DK20541-17 (to Z.-Y. Z.). The purchase of the 500-MHz NMR spectrometer was made possible in part by National Institutes of Health Grant RR02309. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-4288; Fax: 718-829-8705.

The abbreviation used is: PTPases, protein-tyrosine phosphatases.


ACKNOWLEDGEMENTS

I thank Dr. Terry Dowd for assistance with the P NMR data collection. I also thank Drs. John Blanchard and Fred Brewer for comments on the manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.