Site-directed mutagenesis, kinetic, and spectroscopic studies of the P-loop residues in a low molecular weight protein tyrosine phosphatase.

来自 ACS

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作者：

B Evans，PA Tishmack，C Pokalsky，M Zhang，RLV Etten

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摘要：

The structure of the specific phosphate binding loop (P-loop) of bovine protein tyrosine phosphatase (BPTP) is very similar to that present in high Mr 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 ...

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关键词：

Animals Cattle Phosphates Recombinant Proteins Sequence Alignment Circular Dichroism Magnetic Resonance Spectroscopy Conserved Sequence Protein Conformation Protein Denaturation