Investigation of an active site histidine-aspartate couple in Trimeresurus flavoviridis phospholipase A2.
When Trimeresurus flavoviridis phospholipase A2 was reacted with methyl p-nitrobenzenesulfonate, its activity decreased following first-order kinetics. The pH dependence of the rate constants of inactivation showed that His-48 with an apparent pKa of 6.5 controls the reaction. In the pH region below 6.5, N1-methylhistidine was predominantly formed. On the other hand, N1,N3-dimethylhistidine was almost exclusively produced in the pH region above 6.5. No N3-methylhistidine was detected at any pH tested. Such observations suggested that the first methylation occurred at the N1-position of the imidazole ring followed by a second methylation at the N3-position, and that His-48 couples the carboxylate of Asp-99 at the N3-position of the imidazole ring, in accord with the interaction observed in the crystal structure of homologous Crotalus atrox phospholipase A2. As it has been reported that, in the reaction of chymotrypsin with methyl p-nitrobenzenesulfonate at pH 7.8, only monomethylation occurred at the N1-position of the His-57 imidazole group (Nakagawa, Y. & Bender, M.L. (1970) Biochemistry 9, 259-267), the nature of the active site histidine-aspartate couple of T. flavoviridis phospholipase A2 seems not to be identical with that of chymotrypsin.
Kohzuma
T
T
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka.
Oda
N
N
Kihara
H
H
Ohno
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001557:Benzenesulfonates / Q000378:metabolism; D001665:Binding Sites; D003435:Crotalid Venoms; D004789:Enzyme Activation; D006639:Histidine / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008745:Methylation; D008969:Molecular Sequence Data; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D012911:Snakes
D001557:Benzenesulfonates; D003435:Crotalid Venoms; D001224:Aspartic Acid; D006639:Histidine; C010233:methyl-4-nitrobenzenesulfonate; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2