Kinetic, mechanistic, and site-directed mutagenesis studies of mammalian low molecular weight phosphotyrosyl protein phosphatases
Abstract
The recombinant low molecular weight cytoplasmic phosphotyrosyl protein phosphatases (PTPases) from bovine heart (BHPTP) and human placenta (HCPTP-A and -B) were overexpressed in Escherichia coli using a T7 polymerase-dependent bacterial expression system. The enzymes were purified to homogeneity and characterized. All low molecular weight PTPases sequenced to date possess 157 amino acid residues and exhibit a high degree of sequence identity. Site-directed mutagenesis of the recombinant bovine heart enzyme was used to explore the role of the eight conserved cysteines, two conserved histidines, and one (of seven) conserved arginines. Single mutants of cysteine to serine were studied for each cysteine; alanine replacements were also made for the mutants with reduced catalytic activity: Cys-12, Cys-17, and Arg-18. The circular dichroism and $\sp1$H NMR spectra of the purified proteins revealed no apparent conformational structure alterations. Within the limits of measurement, the Cys-12 and Arg-18 mutants were completely inactive. Because concurrent research in this laboratory identified a cysteine as the nucleophilic residue, it is concluded that Cys-12 is the catalytic nucleophile. Arg-18 presumably serves an essential function in substrate binding. Kinetic analysis of C17A indicated that its reduced activity (37% relative to wild type) was not due to a change in the rate-determining step of the catalytic mechanism. Dephosphorylation of the covalent phosphoenzyme intermediate remains rate-limiting. Chemical modification studies using the dithiol modification reagent phenylarsine oxide suggest that Cys-17 is at or near the enzyme active site. Cys-17 may be important for optimal positioning of the substrate phosphate moiety. His-66 and His-72 were individually mutated to alanine and to asparagine. A double mutant, containing only alanines in place of the histidines, was also created. Kinetic studies of the mutant proteins reveal that neither histidine is essential in the catalytic mechanism. His-66 mutants showed virtually identical catalytic properties compared to wild-type enzyme, whereas His-72 mutants had reduced specific activity, higher phosphate K$\sb{\rm i}$ and lower K$\sb{\rm m}$ values at pH 5 and higher. It is proposed that His-72, although not essential for catalysis, may serve a significant structural role at the enzyme active site.
Degree
Ph.D.
Advisors
Etten, Purdue University.
Subject Area
Biochemistry
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