SITE-DIRECTED MUTAGENESIS, KINETIC, AND SPECTROSCOPIC STUDIES OF THE P-LOOP RESIDUES IN A LOW-MOLECULAR-WEIGHT PROTEIN-TYROSINE-PHOSPHATASE

The structure of the specific phosphate binding loop (P-loop) of bovin e protein tyrosine phosphatase (BPTP) is very similar to that present in high M(r) PTPases. Site-directed mutagenesis was used to explore th e role of several conserved residues involved in forming the P-loop of BPTP. Thus, Ser-19 and Ser-43 were individually mutated to alanines, and Asn-15 was mutated to alanine and glutamine. The H-1 NMR spectra o f the mutants showed good conservation of global secondary structure w hen compared to wild-type enzyme. Kinetic measurements revealed that o nly S19A and N15A had substantially altered catalytic activities towar d p-nitrophenyl phosphate at pH 5.0, with both mutants exhibiting V-ma x values that were 0.25-0.33% of wild-type enzyme. Further kinetic ana lyses of the N15A and S19A mutants were performed using phosphomonoest er substrates with varied phenolic leaving groups. For S19A, the slope of the correlation between V-max and the substrate leaving group pK(a ) was significantly altered, consistent with a change of the rate-dete rmining step from dephosphorylation to phosphorylation. This was confi rmed by partitioning experiments employing methanol as an alternative nucleophile in the dephosphorylation step. Thus, mutating Ser-19 to al anine reduced the efficiency of nucleophilic attack by Cys-12. It is c oncluded that Ser-19 acts to facilitate the ionization and orientation of Cys-12 for optimal reaction as a nucleophile and as a leaving grou p. It also appears that Asn-15, Ser-19, His-72, and to a lesser extent Ser-43 serve structural functions that allow the active site to adopt an optimal geometry for phosphate binding. The Asn-15 to Ala mutation appears to disrupt the hydrogen-bonding network, with an accompanying alteration of the geometry of the P-loop. These conclusions are also consistent with changes in the stability of the respective proteins, a s measured by urea denaturation.