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The role of His66 and His72 in the reaction mechanism of bovine liver low-M(r) phosphotyrosine protein phosphatase

, : The role of His66 and His72 in the reaction mechanism of bovine liver low-M(r) phosphotyrosine protein phosphatase. Biochemical Journal 298: 427-433

Site-directed mutagenesis of a synthetic gene coding for low-M, phosphotyrosine protein phosphatase from bovine liver has been carried out. The two histidine residues in the enzyme have been mutated to glutamine; both single and double mutants were produced. The mutated and non-mutated sequences have been expressed in Escherichia coli as fusion proteins, in which the low-M-r phosphotyrosine protein phosphatase was linked to the C-terminal end of the maltose-binding protein. The fusion enzymes were easily purified by single-step affinity chromatography. The mutants were studied for their kinetic properties. Both single mutants showed decreased k-cat values (30 and 7 residual activities for His-66 and His-72 respectively), and alterations of the K-1 values relative to four competitive inhibitors were observed. The kinetic mechanism of p-nitrophenyl phosphate hydrolysis in the presence of both single mutants was determined and compared with that of the non-mutated enzyme. The rate-determining step of the catalytic process of the His-66 fwdarw Gln mutant was the same as that found for non-mutated enzyme, whereas for the His-72 fwdarw Gln mutant, both the kinetic constant of the step that causes the formation of a phosphoenzyme covalent intermediate, and the kinetic constant of the step that causes the dephosphorylation of the enzyme covalent intermediate, determined the k-cat value. This observation was confirmed by phosphoenzyme covalent intermediate trapping experiments. The participation of both histidine residues (His-66 and His-72) at the active site is strongly suggested by the results of diethyl pyrocarbonate inactivation of both single mutants, each containing a single histidine residue. Both mutants are completely inactivated by diethyl pyrocarbonate treatment; the competitive inhibitor P-i protects both mutants from inactivation. The His-66/His-72 double mutant was completely inactive.


PMID: 8135752

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LaJoie, W.J.; Wong, W.H.; Hyman, C.; Austin, E.S., 1969: Cutaneous ischemia from epinephrine ion transfer and counterpressure. Journal of Applied Physiology 27(3): 400-402

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