Kinetic and site-directed mutagenesis studies of the cysteine residues of bovine low molecular weight phosphotyrosyl protein phosphatase

The roles of the 8 conserved cysteines and 1 arginine in the low molecular weight phosphotyrosyl protein phosphatases were investigated using site- directed mutagenesis of the recombinant bovine heart enzyme. Single mutants of cysteine to serine were studied for each cysteine; alanine replacements were also made for Cys-12, Cys-17, and Arg-18. The CD spectra of the purified proteins were effectively superimposable, consistent with the conclusion that no major structural alterations had occurred, but ¹H NMR spectroscopy did reveal some spectral shifts in the aromatic region. Kinetic analysis of the mutant proteins demonstrated that only Cys-12, Cys-17, and Arg-18 had significantly altered catalytic activity toward the substrate p-nitrophenyl phosphate at pH 5. The Cys-12 and Arg-18 mutants were effectively inactive. Thus, it is concluded that Cys-12 is the catalytic nucleophile, and Arg-18 presumably serves an essential function in substrate binding. The C17S mutant had 6% residual activity compared with wild type protein, whereas the C17A mutant had 37% activity. Consistent with the observed activity of the Cys-17 mutant, a covalent phosphocysteine intermediate was trapped and identified by ³¹P NMR. Further kinetic analysis of C17A using several aryl phosphate monoester substrates with different leaving group pK(a) values indicated that no change in the rate-determining step of the catalytic mechanism had occurred, that is, dephosphorylation of the covalent phosphoenzyme intermediate remains rate-limiting. The C17A mutant had a 4-fold higher phosphate K(i) and slightly higher K(m) values for p-nitrophenyl phosphate suggesting that Cys-17 may be important for optimal positioning of the substrate phosphate moiety.