Kinetic mechanism of Clostridium perfringens phospholipase C. Hydrolysis of a thiophosphate analogue of lysophosphatidylcholine.
Biochem J
; 280 ( Pt 2): 407-10, 1991 Dec 01.
Article
en En
| MEDLINE
| ID: mdl-1747114
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Fosfolipasas de Tipo C
/
Lisofosfatidilcolinas
/
Clostridium perfringens
Idioma:
En
Revista:
Biochem J
Año:
1991
Tipo del documento:
Article