title: The effect of histidine modification on the activity of myo-inositol monophosphatase from bovine brain.
abstract: The pH dependence of myo-inositol monophosphatase may indicate a role for histidine residues in the catalytic mechanism (Ganzhorn, A. J., and Chanal, M.-C. (1990) Biochemistry 29, 6065-6071). This possibility was investigated by chemical modification. At pH 6.0 and 25 degrees C, the enzyme was inactivated by diethylpyrocarbonate in a pseudo-first order reaction with a bimolecular rate constant of 0.37 M-1 s-1. Two histidines were modified rapidly with no effect on enzyme activity, while 3 residues were modified at a slower rate corresponding to the rate of inactivation. No noticeable changes in the secondary structure of the enzyme were observed by comparison of circular dichroic spectra before and after modification. Treatment of myo-inositol monophosphatase with diethylpyrocarbonate in the presence of inositol 1-phosphate, Mg2+, and Li+ protected 2 residues from modification and decreased the inactivation rate by about 5-fold. Spectrophotometric analysis, the restoration of enzyme activity by hydroxylamine, and the lack of any inhibitory effect with alkylating agents suggest that inactivation is due solely to modification of histidine. We conclude that a histidine residue is essential for activity and may act as a base catalyst during hydrolysis of the substrate.
authors.affiliation: Marion Merrell Dow Research Institute, Strasbourg, France.
authors.forename: P D
authors.initials: PD
authors.lastname: Pelton
authors.forename: A J
authors.initials: AJ
authors.lastname: Ganzhorn
mesh_terms: D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D006639:Histidine; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
chemical_list: D006639:Histidine; D010744:Phosphoric Monoester Hydrolases; C021822:myo-inositol-1 (or 4)-monophosphatase; D004047:Diethyl Pyrocarbonate