title: Kinetic mechanism of Clostridium perfringens phospholipase C. Hydrolysis of a thiophosphate analogue of lysophosphatidylcholine.
abstract: The hydrolysis of S-[2-(hexadecanoyloxy)ethyl]thiophosphocholine (I), an analogue of lysophosphatidylcholine, by Clostridium perfringens phospholipase C, was followed at pH 7.5, 37 degrees C and I 1.0 (maintained with KCl), in a continuous assay, by monitoring the reduction of 5,5'-dithiobis-(2-nitrobenzoic acid) at 412 nm. Simple saturation kinetics are observed with linear mixed-type slope-intercept effects for the hydrolysis of compound (I) with variable [Ca2+] at fixed concentrations of compound (I) and a simple slope effect as [compound (I)] is varied at fixed concentrations of Ca2+. These data are consistent with a simple ordered rapid-equilibrium mechanism in which Ca2+ binds to the enzyme first followed by substrate. The observed kinetic constants at pH 7.5, 37 degrees C and I 1.0 are K1 = 12.0 mM (Ca2+ dissociation), K2 = 36 microM [compound (I) dissociation] and Vmax. = 552 microM.min-1.mg-1. Alkane diammonium salts inhibit the enzyme by a non-competitive mechanism that involves binding to free enzyme, E.Ca2+ and E.Ca2+.S. The use of the simple micellarized substrate under these conditions allows the determination of kinetic and inhibition constants without complications arising from enzyme-micelle interactions.
authors.affiliation: Department of Chemistry, University of Illinois, Chicago 60680.
authors.forename: P R
authors.initials: PR
authors.lastname: Young
authors.forename: W R
authors.initials: WR
authors.lastname: Snyder
authors.forename: R F
authors.initials: RF
authors.lastname: McMahon
mesh_terms: D003016:Clostridium perfringens / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000378:metabolism*; D008823:Micelles; D010710:Phosphates / Q000378:metabolism; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D008244:Lysophosphatidylcholines; D008823:Micelles; D010710:Phosphates; D010738:Type C Phospholipases; C035638:thiophosphoric acid