Form and Function in Protein Dephosphorylation

In contrast to the catalytic mechanism employed by PPs, the PTPs proceed through a phosphoenzyme intermediate. The enzymatic reaction involves phosphoryl-cysteine intermediate formation after nucleophilic attack of the phosphorus atom of the substrate by the thiolate anion of cysteine (Denu et al. 1996xDenu, J.M, Lohse, D.L, Vijayalakshmi, J, Saper, M.A, and Dixon, J.E. Proc. Natl. Acad. Sci. USA. 1996; 93: 2493–2498Crossref | PubMedSee all ReferencesDenu et al. 1996). The reaction can be represented as a two-step chemical process: phosphoryl transfer to the enzyme accompanied by rapid release of dephosphorylated product (denoted by rate constant k(formation) in Equation 1); and hydrolysis of the thiol-phosphate intermediate concomitant with rapid release of phosphate (denoted by rate constant k(hydrolysis) in Equation 1). To form the catalytically competent complex ES, the enzyme binds and reacts with the dianion of phosphate–containing substrate (Figure 3AFigure 3A). On the enzyme an aspartic acid must be protonated and the nucleophilic cysteine must be unprotonated (thiolate anion) for phosphoryl transfer to the enzyme (3xDenu, J.M, Lohse, D.L, Vijayalakshmi, J, Saper, M.A, and Dixon, J.E. Proc. Natl. Acad. Sci. USA. 1996; 93: 2493–2498Crossref | PubMedSee all References, 20xZhang, Z.-Y. J. Biol. Chem. 1995; 270: 11199–11204Crossref | PubMed | Scopus (86)See all References). In the Michaelis complex (Jia et al. 1995xJia, Z, Barford, D, Flint, A.J, and Tonks, N.K. Science. 1995; 268: 1754–1758Crossref | PubMedSee all ReferencesJia et al. 1995), the three nonbridging oxygens of the phosphoryl group are coordinated by bidentate hydrogen bonds to the guanidinium group of arginine and by the backbone amide N-H groups of the active-site loop (Figure 3AFigure 3A) . Situated directly underneath the phosphoryl group and at the base of the active-site cleft, is the nucleophilic cysteine thiolate anion (shown in green, Figure 3AFigure 3A). The substrate (shown in yellow, Figure 3AFigure 3A) is positioned such that attack of the thiolate is directly in line with the P-O bond and ideally situated for efficient expulsion of the leaving group. To further enhance leaving group expulsion, near the top of the cleft the aspartic acid is positioned to act as a general acid by protonating the leaving group phenolic oxygen. The aspartic acid is found on a separate loop (shown in yellow, Figure 2Figure 2) that was shown to be flexible for both Yersinia PTP (Stuckey et al. 1994xStuckey, J.A, Schubert, H.L, Fauman, E.B, Zhang, Z.-Y, Dixon, J.E, and Saper, M.A. Nature. 1994; 370: 571–575Crossref | PubMed | Scopus (303)See all ReferencesStuckey et al. 1994) and PTP1B (Jia et al. 1995xJia, Z, Barford, D, Flint, A.J, and Tonks, N.K. Science. 1995; 268: 1754–1758Crossref | PubMedSee all ReferencesJia et al. 1995). When the enzyme is complexed with phosphorylated substrates, this loop folds over the active site, bringing the aspartic acid into position for leaving group protonation. In the open conformation, the loop is flipped away from the active site and the aspartic acid is approximately 8–12 A removed from its location in the Michaelis complex (Stuckey et al. 1994xStuckey, J.A, Schubert, H.L, Fauman, E.B, Zhang, Z.-Y, Dixon, J.E, and Saper, M.A. Nature. 1994; 370: 571–575Crossref | PubMed | Scopus (303)See all ReferencesStuckey et al. 1994). It is not yet known whether the analogous loop in VHR is capable of such dramatic movement. In the sulfate-bound complex of VHR, the loop does not cover the active site to the same extent as in the other two PTP crystal structures (Yuvaniyama, et al. 1996xYuvaniyama, J, Denu, J.M, Dixon, J.E, and Saper, M.A. Science. 1996; 272: 1328–1331Crossref | PubMedSee all ReferencesYuvaniyama, et al. 1996).Figure 3Catalytic Mechanism of Protein Tyrosine Phosphatases: Enzyme–Substrate Complex and Phosphoenzyme Intermediate(A) A model of the enzyme-substrate complex of PTP derived from two X-ray crystallographic models: Cys-ser mutant of PTP1B complexed with phosphotyrosine (Jia et al. 1995xJia, Z, Barford, D, Flint, A.J, and Tonks, N.K. Science. 1995; 268: 1754–1758Crossref | PubMedSee all ReferencesJia et al. 1995), and Yersinia PTP complexed with vanadate (Denu et al. 1996xDenu, J.M, Lohse, D.L, Vijayalakshmi, J, Saper, M.A, and Dixon, J.E. Proc. Natl. Acad. Sci. USA. 1996; 93: 2493–2498Crossref | PubMedSee all ReferencesDenu et al. 1996). The backbone atoms of the active site loop from cysteine to arginine are shown as a ball-and-stick model. The phospho-tyrosine substrate (shown in yellow) is bound to the center of the active-site motif with the protonated general acid (Asp) lying within hydrogen bonding distance of the scissile oxygen of the substrate. The dianion of the phosphoryl group is coordinated by the nitrogens of the arginine side chain and by the amide groups of the active site motif. The thiolate anion of cysteine is poised for nucleophilic attack. Individuals atoms are represented as spheres with the following colors: Cα and carbonyl carbons, gray; oxygens, red; nitrogens, blue; phosphorus, magenta; sulfur, green; and hydrogens, orange (not found in original crystal structures). Dashed lines represent hydrogen bonds.(B) A model of the phosphoenzyme intermediate of PTP derived in a similar manner as (A) with the same atom colors as (A). The phosphorus is covalently bound to the Sγ of the cysteine. A water molecule (Wat) is in a position such that aspartic acid can abstract the proton. Figures were drawn using Molscript and Raster3D.View Large Image | View Hi-Res Image | Download PowerPoint SlideTo explore the transition-state structure for catalysis, heavy atom kinetic isotope effects have been measured with PTP1, Yersinia PTP and VHR (Hengge et al. 1996xHengge, A.C, Denu, J.M, and Dixon, J.E. Biochemistry. 1996; 35: 7084–7092Crossref | PubMed | Scopus (61)See all ReferencesHengge et al. 1996). The kinetic isotope effects indicate that P-O bond cleavage is far advanced and there is little bond formation to the nucleophile cysteine during the transition state. It was also demonstrated that proton transfer from aspartic acid to the bridging oxygen is concomitant with P-O cleavage, such that no charge is developed on the leaving group. Consistent with concomitant proton transfer, leaving group pKa values have little effect on the rate of phosphoryl transfer (20xZhang, Z.-Y. J. Biol. Chem. 1995; 270: 11199–11204Crossref | PubMed | Scopus (86)See all References, 3xDenu, J.M, Lohse, D.L, Vijayalakshmi, J, Saper, M.A, and Dixon, J.E. Proc. Natl. Acad. Sci. USA. 1996; 93: 2493–2498Crossref | PubMedSee all References). When aspartic acid is replaced by asparagine, phosphopeptides and phosphotyrosine are 100-fold worse than substrates with good leaving groups such as p-nitrophenylphosphate, a commonly employed artificial substrate. Thus, with physiological substrates, proton donation by aspartic acid is more critical for efficient catalysis than with labile artificial substrates.Once the phosphoenzyme intermediate is formed, it must undergo hydrolysis by water to result in the turnover of the enzyme (Figure 3Figure 3). Activation of the water molecule by a general base would be expected to facilitate hydrolysis. Mutagenic studies with VHR have shown that the conserved aspartic acid enhances intermediate hydrolysis (Denu et al. 1996xDenu, J.M, Lohse, D.L, Vijayalakshmi, J, Saper, M.A, and Dixon, J.E. Proc. Natl. Acad. Sci. USA. 1996; 93: 2493–2498Crossref | PubMedSee all ReferencesDenu et al. 1996), suggesting that the aspartic acid may function as the general base by proton abstraction from a water molecule. Further support for this idea comes from the X-ray structure of Yersinia PTP solved with the inhibitor vanadate covalently bound to cysteine (Denu et al. 1996xDenu, J.M, Lohse, D.L, Vijayalakshmi, J, Saper, M.A, and Dixon, J.E. Proc. Natl. Acad. Sci. USA. 1996; 93: 2493–2498Crossref | PubMedSee all ReferencesDenu et al. 1996). With trigonal bipyramidal geometry, the vanadate mimics the transition for both chemical steps and underscores the importance of conserved arginine and aspartic acid residues. Similar to the ES complex, two of the equatorial oxygens of vanadate are hydrogen bonded to arginine in a bidendate fashion. The aspartic acid makes a hydrogen bond of 2.8 A to the apical oxygen of vanadate, consistent with the role as general acid/base.The recently solved structures of protein phosphatases have greatly advanced our knowledge of the molecular mechanism of catalysis and substrate specificity. Understanding the molecular events of these essential reactions will lead to a better understanding of the specific physiological functions of the protein phosphatases. Also, a detailed description of the inherent differences between and among PTPs and PPs will undoubtedly augment the search for and design of specific phosphatase inhibitors.Because of reference limitations imposed in minireviews, we wish to acknowledge the important contributions made by other investigators whose work we were unable to cite. Reference to this work can be found within the cited publications of many of the selected reading articles.