Abstract:
:The structure of the specific phosphate binding loop (P-loop) of bovine protein tyrosine phosphatase (BPTP) is very similar to that present in high M(r) PTPases. Site-directed mutagenesis was used to explore the 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 1H NMR spectra of the mutants showed good conservation of global secondary structure when compared to wild-type enzyme. Kinetic measurements revealed that only S19A and N15A had substantially altered catalytic activities toward p-nitrophenyl phosphate at pH 5.0, with both mutants exhibiting Vmax values that were 0.25-0.33% of wild-type enzyme. Further kinetic analyses of the N15A and S19A mutants were performed using phosphomonoester substrates with varied phenolic leaving groups. For S19A, the slope of the correlation between Vmax and the substrate leaving group pKa was significantly altered, consistent with a change of the rate-determining step from dephosphorylation to phosphorylation. This was confirmed by partitioning experiments employing methanol as an alternative nucleophile in the dephosphorylation step. Thus, mutating Ser-19 to alanine reduced the efficiency of nucleophilic attack by Cys-12. It is concluded that Ser-19 acts to facilitate the ionization and orientation of Cys-12 for optimal reaction as a nucleophile and as a leaving group. 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, as measured by urea denaturation.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Evans B,Tishmack PA,Pokalsky C,Zhang M,Van Etten RLdoi
10.1021/bi9605651subject
Has Abstractpub_date
1996-10-22 00:00:00pages
13609-17issue
42eissn
0006-2960issn
1520-4995pii
bi9605651journal_volume
35pub_type
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