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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.

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PMID: 8135752


Other references

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

Riff, L.J., 1975: Bacteremia arising from the urinary tract. Urologic Clinics of North America 2(3): 521-535

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Arimoto, Y.H.mma, Y., 1986: Studies on citrus melanose and citrus stem-end rot by Diaporthe citri (Faw.) Wolf. 6. Cell-division inducer produced in melanose spot. Nihon Shokubutsu Byori Gakkaiho = Annals of the Phytopathological Society of Japan 52(4): 683-689

Deloule E.; France Lanord Christian; Albarede F., 1991: D/ H analysis of minerals by ion probe. Special Publication - Geochemical Society 3: 53-62

Gans R.O.B.; Kuizinga M.C.; Huysmans F.; Gerlag P.G.G.; Hoorntje S.J., 1989: Favorable outcome of rapidly progressive glomerulonephritis rpgn in systemic vasculitis with anti neutrophil cytoplasmic autoantibodies anca. Kidney International 35(1): 226

Beachley, M.C.; Franklin, J.W.; Ostlund, W.; Fielding, E.; Lenkey, J.; Urbanik, E., 1993: Radiology of arthritis. The radiographic evaluation of the arthritides still relies primarily on the plain films that are readily available to every practicing clinician. The radiographic examination serves primarily to confirm the clinician's diagnosis based on the...

Roongpisuthipong, A.; Siriwasin, W.; Chaiyakul, P.; Bhiraleus, P.; Laosakkitiboran, J.; Shaffer, N.; Mastro, T.D., 1994: Husband HIV discordance among HIV-infected pregnant women, Bangkok, Thailand. TENTH INTERNATIONAL CONFERENCE ON AIDS, INTERNATIONAL CONFERENCE ON STD [Author] Tenth International Conference on AIDS and the International Conference on STD, Vol 1; The global challenge of AIDS: Together for the future : 1) 32