title: Mechanistic studies on full length and the catalytic domain of the tandem SH2 domain-containing protein tyrosine phosphatase: analysis of phosphoenzyme levels and Vmax stimulatory effects of glycerol and of a phosphotyrosyl peptide ligand.
abstract: SHP-1, a protein tyrosine phosphatase containing two tandem SH2 domains, is autoinhibited at rest by its N-terminal SH2 (N-SH2) domain. Relief from autoinhibition and a subsequent 10-60-fold increase in V(max) have been observed upon N-SH2 domain engagement by a specific phosphotyrosyl ligand or upon deletion of the SH2 domains to yield the catalytic PTPase domain. In this study, we observed that glycerol and propane-1,2-diols, at concentrations of 4-6 M, accelerated the k(cat) of the full length enzyme by 47-fold and of the PTPase domain by 8-fold. Glycerol also increases the rate of proteolytic cleavage between the SH2 and catalytic PTPase domains. In stopped-flow studies using p-nitrophenyl phosphate (pNPP) as a substrate, a burst of p-nitrophenolate in the full length enzyme was not observed; however, a 50-70% stoichiometric burst was observed with the PTPase domain. Rapid quench studies using [32P]pNPP as a substrate showed a very low level of covalent [32P]phosphocysteinyl enzyme intermediate accumulation: 0.06% in the full length enzyme and 1% in the PTP domain. Stimulation by glycerol reduced the accumulating levels of phosphocysteinyl enzyme in both cases of full length SHP-1 and the PTPase domain; however, glycerol is not acting as a cosubstrate since no glycerophosphate product was detectable. It is likely that, for full length SHP-1, with pNPP as a model substrate, enzyme-substrate complex (ES) accumulates in its basal autoinhibited state, whereas enzyme-product complex (EP(i)) accumulates in its pY ligand-bound activated state. Glycerol probably relaxes the compact structure of SHP-1 and the PTP domain, thereby accelerating the catalytic rates in both cases by increasing forward reaction rates of ES and EP(i).
authors.affiliation: Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: C T
authors.initials: CT
authors.lastname: Walsh
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine / Q000031:analogs & derivatives; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005990:Glycerol / Q000494:pharmacology*; D006868:Hydrolysis; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010196:Pancreatic Elastase / Q000378:metabolism; D010748:Phosphopeptides / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D011409:Propylene Glycols / Q000494:pharmacology*; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D018909:src Homology Domains
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010748:Phosphopeptides; D011409:Propylene Glycols; C008644:nitrophenylphosphate; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D010196:Pancreatic Elastase; D003545:Cysteine; D005990:Glycerol