Mechanistic, kinetic, and chemical modification studies of low molecular weight protein tyrosine phosphatase

Zhongtao Zhang, Purdue University

Abstract

The kinetic parameters of low Mr PTPase were characterized. The enzyme was confirmed to use a cysteine residue as a nucleophile for the catalysis of phosphomonoester hydrolysis with trapping experiments. A new scheme was devised to further characterize the nucleophilic residue which was directly identified as cysteine 12. Two small acids, tartronic acid and mesoxalic acid, were proposed to mimic the transition-state of the enzyme-catalyzed hydrolysis of phosphomonoesters. Both compounds inhibit strongly the low Mr PTPases and prostatic acid phosphatase. Dicarboxylates, such as succinate, were found to bind low Mr PTPase and stimulate the enzymatic activity by reducing the apparent K$\rm\sb m$ values for the substrate. The binding of a dicarboxylate also increased the inactivation rate of the enzyme by covalent modification reagents directed to the active site. A working model of the dicarboxylate effect was proposed. The dicarboxylate is proposed to mimic the acidic residues which are at the N-terminal of the phosphotyrosine to the possible physiological substrate. The binding of the dicarboxylate makes the active site more accessible to the modification reagents. Photo cross-linking studies were carried out on the low Mr PTPase. A new method for the synthesis of the photo crosslinking reagent was designed and carried out. The photo crosslinking results indicated that cysteine 12 is close to cysteine 17 and cysteine 17 is close to tyrosine 131. Together with sequence alignment analysis, the structure of the low Mr PTPase was proposed to share certain structural motifs with adenylate kinase. All these proposals were later confirmed by the crystal structure of the bovine low Mr PTPase. Aspartic acid residue 129 was identified as the proton donor to the leaving group in the phosphorylation step by site-directed mutagenesis and characterization of the mutant enzymes. The mutations of D129A and D129E both resulted in the slow down of the phosphorylation step and render it rate limiting. Extensive leaving group dependence studies and solvent isotope effect studies were carried out on several mutant bovine low Mr PTPases. The results indicate that the nucleophilic residue C12 has an abnormally low pK$\rm\sb a$ of 4.25. To explain all the kinetic results, a new catalytic mechanism involving a shift of the phosphoenzyme intermediate was proposed.

Degree

Ph.D.

Advisors

Etten, Purdue University.

Subject Area

Biochemistry|Organic chemistry|Pharmacology

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