Mechanistic, kinetic, and spectroscopic studies on low molecular weight tyrosine phosphatases

Christine N Pokalsky, Purdue University

Abstract

The structural and mechanistic roles of several residues of low $M\sb{\rm r}$ PTPase were investigated by a variety of techniques including, but not limited to, site-directed mutagenesis, fluorescence quenching via steady state and lifetime techniques, urea denaturation, phosphoenzyme trapping, transient and steady state kinetics, and pH titrations. In the bovine protein tyrosine phosphatase (BPTP) Trp$\sp{39}$ and Trp$\sp{49}$ were found to have important roles in affecting the protein stability and substrate binding, respectively. Adenine, an effector of BPTP activity, and pNPP, an artificial substrate of BPTP, both directly interact with Trp$\sp{49},$ quenching fluorescence via a static mechanism. The pK$\rm\sb{a}$ of Cys$\sp{12}$ was found to be directly dependent on the influence of residues, particularly Ser$\sp{19},$ located within the microenvironment of the active site. Additionally, as deduced from the pH profiles of BPTP and mutant proteins of BPTP, the active form of the enzyme consists of Cys$\sp{12}$ as an anionic species and a proton donor, Asp$\sp{129}.$ The artificial substrate, pNPP, exists in a dianionic state in the enzyme-substrate complex. Use of the mutant proteins D129E and D129A BPTP clarify the role of Asp$\sp{129}$ as the proton donor. Transient kinetic studies clearly show that hydrolysis of pNPP by D129A BPTP is limited by the rate of phosphorylation of the enzyme. Previous results presented indicated that D129E BPTP also displays rate-limiting phosphorylation. However, transient kinetic studies done as a function of pH, clearly show regions of pH in which D129E BPTP displays only partial rate determining phosphorylation. The structural significance of Ser$\sp{19},$ as investigated by urea denaturation curves, indicates the potential loss of at least one hydrogen bond, consistent with the proposed theory that Ser$\sp{19}$ hydrogen bonds to Cys$\sp{12}.$ Geometry optimization computations show that mutation of Ser$\sp{19}$ to Ala results in a slight alteration in backbone structure with Ala located 2.12 A further from the nucleophilic cysteine residue. Experimental measurements show that the mutant BPTP proteins H72A, N15A, and S43A all display a loss of protein stability, consistent with the presence of a critical hydrogen-bonding network located beyond the immediate active site.

Degree

Ph.D.

Advisors

Etten, Purdue University.

Subject Area

Biochemistry|Analytical chemistry

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