Mechanistic and kinetic studies of the bovine heart low molecular weight phosphotyrosyl protein phosphatase

Zhong-Yin Zhang, Purdue University

Abstract

The low molecular weight phosphotyrosyl protein phosphatase from bovine heart was purified to homogeneity and extensively characterized. Substrate specificity studies revealed that the enzyme was active towards aryl phosphate monoesters (with the exception of ortho-substituted derivatives), flavin mononucleotide, and its structural analogues. Guanidine hydrochloride caused a two-stage reversible denaturation of the enzyme with midpoints of 0.25 and 1.75 M respectively. The pH-dependence of the enzyme catalyzed hydrolysis reaction suggested that four residues with pKa's of 3.5-4.0, 6.2, 7.2 and 8.5 were important for enzyme action, and the k$\sb{\rm cat}$ was constant from pH 3.5 to 7.0. The existence of a covalent phosphoenzyme intermediate was strongly implicated by a variety of kinetic experiments, including burst titration, isotope exchange reaction, partition experiments, effects of phosphate acceptor on the steady state kinetic parameters, and product inhibition study. The rate-limiting step for the enzyme catalyzed aryl phosphate hydrolysis was shown to be the breakdown of the phosphoenzyme intermediate. An energetic diagram was constructed for the enzyme catalyzed reaction. Leaving group dependence of the enzyme phosphorylation demonstrated a strong electrophilic participation in the transition state of the enzyme catalyzed reaction. Proton inventory experiments indicated that only one solvent derived proton was "in-flight" in the transition state and that this proton transfer was responsible for the reduction of the effective charge on the departure oxygen atom. Chemical modification experiments suggested that at least one Arg, one His (pKa = 5.5), and two Cys residues (pKa = 7.2 and 8.6, respectively) were required for enzyme activity. Cysteine modification experiments revealed a differential reagent reactivity and a D$\sb2$O solvent sensitivity for the enzyme inactivation. The identity of two essential Cys residues was found to be Cys-62 and Cys-145 in the protein sequence, using an active-site directed inactivator. $\sp{32}$P-labeled substrate was used in trapping experiments and the enzyme was labeled with a 74% stoichiometry. Chemical stability studies of the phosphoenzyme intermediate showed that the nucleophilic residue being labeled was most likely a Cys residue. A unified model for the active site structure and mechanism was proposed.

Degree

Ph.D.

Advisors

Etten, Purdue University.

Subject Area

Biochemistry

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