Functional Characterization and Crystal Structure of the C215D Mutant of Protein-tyrosine Phosphatase-1B

We have characterized the C215D active-site mutant of protein-tyrosine phosphatase-1B (PTP-1B) and solved the crystal structure of the catalytic domain of the apoenzyme to a resolution of 1.6 Å. The mutant enzyme displayed maximal catalytic activity at pH ∼4.5, which is significantly lower than the pH optimum of 6 for wild-type PTP-1B. Although both forms of the enzyme exhibited identical K m values for hydrolysis of p-nitrophenyl phosphate at pH 4.5 and 6, the k cat values of C215D were ∼70- and ∼7000-fold lower than those of wild-type PTP-1B, respectively. Arrhenius plots revealed that the mutant and wild-type enzymes displayed activation energies of 61 ± 1 and 18 ± 2 kJ/mol, respectively, at their pH optima. Unlike wild-type PTP-1B, C215D-mediated p-nitrophenyl phosphate hydrolysis was inactivated by 1,2-epoxy-3-(p-nitrophenoxy)propane, suggesting a direct involvement of Asp215 in catalysis. Increasing solvent microviscosity with sucrose (up to 40% (w/v)) caused a significant decrease in k cat/K m of the wild-type enzyme, but did not alter the catalytic efficiency of the mutant protein. Structurally, the apoenzyme was identical to wild-type PTP-1B, aside from the flexible WPD loop region, which was in both “open” and “closed” conformations. At physiological pH, the C215D mutant of PTP-1B should be an effective substrate-trapping mutant that can be used to identify cellular substrates of PTP-1B. In addition, because of its insensitivity to oxidation, this mutant may be used for screening fermentation broth and other natural products to identify inhibitors of PTP-1B. We have characterized the C215D active-site mutant of protein-tyrosine phosphatase-1B (PTP-1B) and solved the crystal structure of the catalytic domain of the apoenzyme to a resolution of 1.6 Å. The mutant enzyme displayed maximal catalytic activity at pH ∼4.5, which is significantly lower than the pH optimum of 6 for wild-type PTP-1B. Although both forms of the enzyme exhibited identical K m values for hydrolysis of p-nitrophenyl phosphate at pH 4.5 and 6, the k cat values of C215D were ∼70- and ∼7000-fold lower than those of wild-type PTP-1B, respectively. Arrhenius plots revealed that the mutant and wild-type enzymes displayed activation energies of 61 ± 1 and 18 ± 2 kJ/mol, respectively, at their pH optima. Unlike wild-type PTP-1B, C215D-mediated p-nitrophenyl phosphate hydrolysis was inactivated by 1,2-epoxy-3-(p-nitrophenoxy)propane, suggesting a direct involvement of Asp215 in catalysis. Increasing solvent microviscosity with sucrose (up to 40% (w/v)) caused a significant decrease in k cat/K m of the wild-type enzyme, but did not alter the catalytic efficiency of the mutant protein. Structurally, the apoenzyme was identical to wild-type PTP-1B, aside from the flexible WPD loop region, which was in both “open” and “closed” conformations. At physiological pH, the C215D mutant of PTP-1B should be an effective substrate-trapping mutant that can be used to identify cellular substrates of PTP-1B. In addition, because of its insensitivity to oxidation, this mutant may be used for screening fermentation broth and other natural products to identify inhibitors of PTP-1B. The protein-tyrosine phosphatases (PTPases) 1The abbreviations used are: PTPases, protein-tyrosine phosphatases; PTP-1B, protein-tyrosine phosphatase-1B; MES, 4-morpholineethanesulfonic acid; pNPP, p-nitrophenyl phosphate; EPNP, 1,2-epoxy-3-(p-nitrophenoxy)propane. compose a family of ∼100 enzymes that play an important role in controlling several biological processes, including cell cycles and signal transduction pathways (1van Huijsduijnen R.H. Gene (Amst.). 1998; 225: 1-8Crossref PubMed Scopus (67) Google Scholar, 2Zhang Z.-Y. Crit. Rev. Biochem. Mol. Biol. 1998; 33: 1-52Crossref PubMed Scopus (252) Google Scholar). One member of this family of phosphatases that is receiving increased attention because of its potential role in controlling the insulin signaling pathway is protein-tyrosine phosphatase-1B (PTP-1B). Studies have suggested that this ubiquitously expressed enzyme may play a role in regulating the function of the insulin receptor. Thus, PTP-1B is an attractive target for drug design in the treatment of Type II diabetes (for reviews, see Refs. 3Kennedy B.P. Ramachandran C. Biochem. Pharmacol. 2000; 60: 877-883Crossref PubMed Scopus (134) Google Scholar and 4Ukkola O. Santaniemi M. J. Intern. Med. 2002; 251: 467-475Crossref PubMed Scopus (81) Google Scholar). Like other PTPases, PTP-1B contains a conserved 11-residue sequence motif (i.e. (I/V)HCXAGXXR(S/T)G) that harbors Cys215, which acts as the nucleophile and is essential for catalysis. The signature motif also forms the “P-loop” that is involved in substrate binding and catalysis. It is thought that PTP-1B-mediated catalysis occurs via a double-displacement mechanism in which the phosphoryl group of the substrate is first transferred to the active-site Cys residue (Cys215) (5Guan K.L. Dixon J.E. J. Biol. Chem. 1991; 266: 17026-17030Abstract Full Text PDF PubMed Google Scholar, 6Cho H. Krishnaraj R. Bannwarth W. Walsh C.T. Anderson K.S. J. Am. Chem. Soc. 1992; 114: 7296-7298Crossref Scopus (112) Google Scholar). The initial phosphoryl transfer is assisted by an invariant Asp residue (Asp181) residing in a flexible loop region (WPD loop) that spans the conserved tripeptide Trp-Pro-Asp. It is generally believed that Asp181 first acts as a general acid and protonates the leaving group in the phosphorylation step. Subsequently, Asp181 functions as a general base, abstracting a proton from an attacking water molecule in the dephosphorylation step to enhance the rate of hydrolysis of the enzyme-thiophosphate intermediate (7Zhang Z.-Y. Wang Y. Wu L. Fauman E. Stuckey J.A. Schubert H.L. Saper M.A. Dixon J.E. Biochemistry. 1994; 33: 15266-15270Crossref PubMed Scopus (172) Google Scholar, 8Hengge A.C. Sowa G. Wu L. Zhang Z.-Y. Biochemistry. 1995; 34: 13982-13987Crossref PubMed Scopus (124) Google Scholar, 9Wu L. Zhang Z.-Y. Biochemistry. 1996; 35: 5426-5434Crossref PubMed Scopus (63) Google Scholar, 10Denu J.M. Lohse D.L. Vijayalakshmi J. Saper M.A. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2493-2498Crossref PubMed Scopus (252) Google Scholar). Previous structural studies on PTP-1B have revealed interesting details regarding the conformations and structural organizations of the WPD loop and P-loop regions. Specifically, the WPD loop has been shown to adopt different conformations in the unliganded and liganded forms of the enzyme. In the unliganded structure, the WPD loop is in an open conformation, in which Asp181 is ∼10 Å away from the P-loop. Upon substrate binding, the WPD loop adopts a closed conformation and covers the active site like a “flap,” thereby positioning Asp181 closer to the leaving group (11Barford D. Flint A.J. Tonks N.K. Science. 1994; 263: 1397-1404Crossref PubMed Scopus (688) Google Scholar, 12Jia Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Crossref PubMed Scopus (559) Google Scholar). In wild-type PTP-1B, Cys215 is present as a thiolate (13Dillet V. Van Etten R.L. Bashford D. J. Phys. Chem. 2000; 104: 11321-11333Crossref Scopus (50) Google Scholar), and it is known that this active-site residue is absolutely necessary for PTP-1B-mediated catalysis. Mutation of this residue to a neutral Ser generates a “substrate-trapping” mutant, which is able to bind substrates with affinities similar to those of the wild-type enzyme, but does not display any measurable phosphatase activity (14Zhang Z.-Y. Wu L. Biochemistry. 1997; 36: 1362-1369Crossref PubMed Scopus (29) Google Scholar, 15Skorey K.I. Kennedy B.P. Friesen R.W. Ramachandran C. Anal. Biochem. 2001; 291: 269-278Crossref PubMed Scopus (21) Google Scholar). The crystal structure of the unliganded C215S PTP-1B mutant shows the P-loop in a conformationally distinct orientation compared with that found in the wild-type protein. However, in the liganded form, the P-loop adopts the same conformation as the wild-type protein. In the C215S mutant, substitution of the negatively charged thiolate with a neutral (although polar) alcohol destabilizes the PTPase signature motif loop (P-loop) and the surrounding areas, favoring the extended conformation (16Scapin G. Patel S. Patel V. Kennedy B. Asante-Appiah E. Protein Sci. 2001; 10: 1596-1605Crossref PubMed Scopus (36) Google Scholar). The structural studies therefore suggest that the conformation of the P-loop region of the enzyme is inducible and may be dependent on the presence of the negative charge of the active-site nucleophile. The goal of this study was to explore the importance of the presence of a negatively charged residue other than Cys at position 215 in the conformation of the P-loop and in the catalytic activity of PTP-1B. We therefore substituted Cys215 with Asp, as this residue is similar in charge and size density to the active-site thiolate. Here, we report the functional characterization and crystal structure of the C215D mutant enzyme and compare with those of wild-type PTP-1B. and the were from were from and were from and inhibitors were from Science. The and were from other were from were by a of the as a Anal. Chem. Scholar). and of mutant and wild-type PTP-1B, were in for and on The were transferred at a of for The was with an and by The was used for of the suggested and Protein the catalytic domain of PTP-1B in a was used as a for The was a R. in Scholar). The was by on an and the sequence were the The was E. for The were in broth at with at to an of The were by the of 1 and for an 2 were by and cell were by and the of and of mutant and wild-type PTP-1B was as E. Friesen R. C. C. R. G. Ramachandran C. Kennedy B.P. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). E. with the were in of and of of at a pH of was by an cell at The was of the cell at for and to a with inhibitors of the the was with of binding the at to The was with a of in binding were by the by and also by PTPase The were and the was to The was to a with and at pH The was with binding to the at to the was with a of The were by and by enzyme the were The was and at pH to at in the were at on with the of a a rate of 1 was used in were in a at in of MES, 2 and at the The of this in a pH of PubMed Scopus Google Scholar). activity was by C215D wild-type hydrolysis of p-nitrophenyl phosphate to hydrolysis was by C215D wild-type PTP-1B with for respectively, and the by to a of 1 The at was on a and the catalytic activity was the of the The of were to the the to values from the the were also of Arrhenius C215D and wild-type PTP-1B were at their pH at as a substrate and an of respectively. The were Arrhenius and the of the were with the the was in and to the to a of of the enzymes for 1 at in the presence of EPNP, the catalytic were at their pH as of on C215D wild-type PTP-1B activity was by hydrolysis the in of sucrose were at the in 4.5 and to the to a The were the to of the solvent the the in were the at Z. D. D. M. Scholar, M. J. and and Google Scholar). The used in were and for and 40% sucrose respectively. and were by in at by 2 of in 1 and pH and 2 of and pH were on an from a crystal in to 1.6 Å were at in the of the at the and were with the Biol. PubMed Scopus Google Scholar, Scholar). The crystal was with group and cell a and for the for the and crystal was to PTP-1B (i.e. Protein Y. M. Zhang Z.-Y. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar), and the structure of the C215D mutant was solved by as the initial the structure of the mutant enzyme in with solvent and (WPD loop) and (P-loop) were from the The initial density to Å from this that the WPD loop was in a closed conformation and that the the catalytic site the same conformation in the wild-type enzyme were the density the M. 1997; PubMed Scopus Google Scholar). of the was by cycles of of the in and including to cycles of and and were in solvent was the and the was the maximal A. 1996; Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). resolution were it in the density that the WPD loop was present in both “open” and “closed” conformations. other were also as conformation for their the for in conformations was to for and to the At the of the cycles of were for the in the for the for the the importance of a negatively charged residue at position 215 of PTP-1B, we substituted Cys215 with The of this acid the of structural in the conformation of the P-loop. and of the C215D mutant, the functional and structural of the enzyme were compared with those of wild-type PTP-1B. of pH on C215D the pH of C215D and wild-type hydrolysis of at pH Although the pH were generally for both suggesting the of acid involved in significant in the were wild-type PTP-1B displayed maximal catalytic activity at pH the active-site Cys was with Asp, the pH optimum of the enzyme was to the pH and significant in the of catalytic activity were at the pH of the at pH the C215D mutant displayed maximal the catalytic activity of wild-type PTP-1B was of its wild-type PTP-1B exhibited maximal catalytic activity at pH 6, of the maximal catalytic activity of the C215D mutant was the pH the first and of the (i.e. and for wild-type PTP-1B were to be and respectively. to the of the acid of the pH for the C215D mutant, the not be and it be that the was and to the pH the of the mutant enzyme was also significantly lower than that of wild-type PTP-1B. that the forms of the enzyme displayed distinct pH with to substrate of K m k cat and of and we compared the of the C215D mutant with those of wild-type PTP-1B. shows a of the of C215D and wild-type PTP-1B-mediated hydrolysis at the pH optima. Although both enzymes displayed similar K m values of the k cat values of the C215D mutant were ∼70- and ∼7000-fold lower than those of wild-type PTP-1B at pH 4.5 and 6, of C215D and wild-type PTP-1B-mediated hydrolysis at pH 4.5 and ± ± ± ± ± ± ± ± in a the in k cat the mutant and wild-type enzymes be at by potential in the of we compared the activation energies of C215D and wild-type hydrolysis by the catalytic as a function of The Arrhenius plots and the values for C215D and wild-type PTP-1B shown in 2 and respectively. The Arrhenius for wild-type PTP-1B-mediated catalysis was the of with 18 ± 2 At significant of the enzyme was not The C215D mutant was and a Arrhenius was to this the Arrhenius also to to of the enzyme. the for the C215D mutant (i.e. 61 ± 1 was than that for wild-type energies for hydrolysis by C215D and wild-type ± ± 2 in a of C215D-mediated by is a of enzymes that an active-site residue (for see Refs. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R. Biochemistry. PubMed Scopus Google Scholar, R. J. Z. A. Z. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). is to of the active-site residue by the of the C215D mutant contains a residue in its active we to the of on the catalytic activity of this mutant of PTP-1B. We therefore the of C215D and wild-type PTP-1B catalytic that a of the enzymes at in the presence of The activity of the mutant was by of the enzyme in the presence of the However, the activity of wild-type PTP-1B was not significantly similar the acid the mutant and wild-type enzymes was the substitution of the catalytic Cys215 with Asp in the we that inactivated C215D of the group of of the the of the substitution on the structure of PTP-1B, we solved the crystal structure of the mutant enzyme and it The a of is for and 1.6 Å for and for and of Å and The conformation of of the was the of the Ramachandran with in as J.M. J. Google Scholar). The of and were as conformations. and the WPD loop) and solvent were also as conformations. The P-loop is in the wild-type conformation and present the Asp215 and the and of and and the of as in Asp215 is also to of several water in the binding water is at the position by of the phosphate of in the Protein structure and similar with the of and water molecule the position of phosphate but to be and it has been as distinct water molecule with the and of the of and water The other solvent at of which to be have been in the binding an Asp215 to Asp181 in the WPD loop and The WPD loop both the active closed conformation with an of and an of and the open conformation of the unliganded enzyme. position to a lower on with an of but density was in the that be by a open the different of the WPD the of also distinct conformations similar that to the conformations in the and liganded PTP-1B and for the used to the structure of the mutant of PTP-1B and for the of of of were aside for of were aside for of of to of solvent of and have been in the of were aside for to and have been in the in a of on C215D and PTP-1B-mediated the structural the wild-type enzyme and the C215D was in the conformation of the WPD we to this we the of loop to C215D and wild-type PTP-1B-mediated catalysis by the of solvent We the K m and k cat of hydrolysis of in the presence of of shown in the k cat of the wild-type enzyme was in the presence of 40% However, was a decrease in k cat/K m of wild-type PTP-1B suggesting that the catalytic efficiency of wild-type PTP-1B was significantly in the presence of In was in the K m k cat of C215D-mediated catalysis similar in a of of cat/K cat/K as a function of solvent microviscosity and we that substitution of Cys215 with Ser in a conformationally distinct P-loop in the unliganded mutant enzyme, and we suggested that a negative charge in the active site is to the P-loop conformation in the wild-type enzyme (16Scapin G. Patel S. Patel V. Kennedy B. Asante-Appiah E. Protein Sci. 2001; 10: 1596-1605Crossref PubMed Scopus (36) Google Scholar). In this we and characterized the C215D mutant of PTP-1B to the structural of PTP-1B catalysis. We substituted Cys215 with Asp to both the charge and size of this active-site of the active-site Cys with Asp in a in the pH for hydrolysis of wild-type PTP-1B displayed a pH optimum of with the optimum values of for the enzyme N.K. J. Biol. Chem. 263: Full Text PDF PubMed Google Scholar, E. D. A. R. J. Biochem. 1994; PubMed Scopus Google Scholar). In the pH optimum for the C215D mutant was to a significantly lower of and the that the active-site which a a of in wild-type PTP-1B (13Dillet V. Van Etten R.L. Bashford D. J. Phys. Chem. 2000; 104: 11321-11333Crossref Scopus (50) Google Scholar), was with the Asp with a a of in The lower pH optimum of the C215D is similar to the pH of enzymes that active-site Asp as J. J. Biol. Chem. Full Text PDF PubMed Google and the R. Protein 1994; PubMed Scopus Google Scholar). both forms of PTP-1B displayed identical K m values at the pH optimum of the C215D The K m values of and for for both mutant and wild-type PTP-1B, respectively, were than the K m values at pH both displayed the same in K m at a lower pH that the in K m was not a of substitution of the active-site Cys with the in K m may be to the of pH on the of the substrate may be a of in the of other important in the of the active-site of the At pH C215D-mediated hydrolysis lower than wild-type hydrolysis at the same Thus, substitution of the active-site Cys with negatively charged Asp caused a significant in the rate of substrate that not be to that of the wild-type enzyme at the pH optimum of the mutant protein. The Arrhenius plots for C215D and wild-type PTP-1B revealed that the activation of C215D-mediated hydrolysis was than that of the by wild-type PTP-1B. The plots were of for the C215D mutant and for the wild-type enzyme and did not any the Thus, the in k cat for the mutant with to hydrolysis may be to the activation of C215D-mediated catalysis. The crystal structure of the mutant shows several water at the active water have to be from the active site to substrate binding and catalysis. Thus, we that the that from the of the active site to the activation for the mutant protein. is a of enzymes that an active-site residue and has been used to study the of as J. J. Biol. Chem. Full Text PDF PubMed Google and the and (for see Refs. R. J. Z. A. Z. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar and Z. A. J. Am. Chem. Soc. 1996; Scopus Google Scholar). by this molecule is to of the active-site residue by the of of the C215D by and the of an on wild-type PTP-1B suggest a direct involvement of Asp215 in the catalytic mechanism of the mutant protein. Zhang Z.-Y. Van Etten R.L. Biochemistry. 1992; PubMed Scopus Google that also acts as an of the PTPase from In the of this were to be the target of the did not significantly wild-type PTP-1B, it to suggest that the of the C215D mutant was a of a of the active-site Asp by the as for the pH studies and that Asp215 is to the mechanism of the mutant protein. Structurally, wild-type PTP-1B and the C215D with the of the WPD which in both open and closed conformations in the mutant protein. The closed conformation is the open conformation of and and is by the both solvent and in the binding site in the flexible WPD loop to solvent microviscosity the catalytic of of the WPD loop is to the catalytic mechanism of PTP-1B, it is that solvent microviscosity have a on PTP-1B-mediated catalysis by a to the of this The catalytic domain of wild-type PTP-1B was by solvent in a decrease in k cat/K m in the presence of 40% from a K m In addition, we have similar studies on the PTPase and have found that the catalytic efficiency of the region of this enzyme (i.e. to in solvent microviscosity in a similar to PTP-1B not Specifically, k cat/K m of hydrolysis was lower in the presence of 40% The decrease in catalytic efficiency in the presence of sucrose is with the that of the WPD loop is to the catalytic mechanism of PTP-1B. However, the to the same did not the K m k cat of the C215D mutant and in in the rate of C215D-mediated of the crystal of and wild-type PTP-1B an the of solvent on catalytic The closed conformation necessary for catalysis is in the structure, but not in the wild-type that is a for the loop to the conformation in the mutant in of substrate than in the wild-type enzyme. It that this structural in the WPD loop conformation in the C215D mutant, which in a active the of to the solvent mechanism may the K m for the wild-type enzyme in the presence of the important in the of potential substrates of is the of substrate-trapping that similar to the wild-type enzyme, but display a lower the enzyme and the substrate a substrate of substrate-trapping have been used to PTP-1B. In the first the active-site Cys is with Ser Dixon J.E. S. J. Med. 1992; PubMed Scopus Google Scholar, H. Tonks N.K. Full Text PDF PubMed Scopus Google Scholar, K.L. G. D. A. Zhang Z.-Y. Dixon J.E. J. Biol. Chem. 268: Full Text PDF PubMed Google Scholar). mutant the to bind but measurable catalytic However, in the for binding the of the C215S mutant and wild-type PTP-1B have been M. Wu L. Zhang Z.-Y. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google and be by the conformation of the P-loop in the mutant enzyme (16Scapin G. Patel S. Patel V. Kennedy B. Asante-Appiah E. Protein Sci. 2001; 10: 1596-1605Crossref PubMed Scopus (36) Google Scholar). In the of substrate-trapping mutant, the general acid Asp (i.e. is with A.J. Flint A.J. Tonks N.K. Mol. Biol. 1996; PubMed Scopus Google Scholar, A.J. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). Like the C215S mutant, this enzyme also but its catalytic activity is A.J. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, Z.-Y. Wang Y. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). of substrate-trapping mutant is the which has been used to a crystal structure of the intermediate Flint A.J. Tonks N.K. Barford D. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). a mutant of PTP-1B has been substrate-trapping mutant than both the and C215S for the and and lower k cat values for and L. Zhang Zhang Z.-Y. Biochemistry. 2002; PubMed Scopus (81) Google Scholar). It is this mutant with wild-type PTP-1B. In wild-type PTP-1B, Cys215 functions as a a intermediate in the We that substitution of this residue with negatively charged Asp decrease the catalytic activity of the enzyme. We that the of the C215D mutant was significantly lower than that of the wild-type at pH 6, suggesting that this PTP-1B may also as a substrate-trapping of the enzyme. In this we have characterized of the functional of the C215D of PTP-1B. Although the of this study suggest that Asp215 is involved in it is the catalytic mechanism of this is identical to that of the wild-type enzyme is in to this the C215D enzyme is a substrate-trapping mutant structure is identical but significantly lower catalytic activity wild-type PTP-1B. Thus, C215D be used to and identify physiological substrates of PTP-1B. of a residue in of a thiolate has the enzyme to and this mutant may also be used for screening of fermentation broth and natural products to identify inhibitors of PTP-1B. studies in that the of inhibitors with the mutant enzyme to those with the wild-type enzyme. We the at the of the for

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