A steady state analysis of pH and temperature effects on the action of an arginine esterase from the venom of the gabon adder, Bitis gabonica, on synthetic arginine substrate.

Abstract

The influence of pH and temperature on the kinetic properties of esterase E-II from Bitis gabonica venom has been studied. The pH profiles for the hydrolysis of N alpha-benzoyl-L-arginine ethyl ester showed that a group with pK approximately 7 must be ionized for activity. Measurement of N alpha-benzyl-L-arginine-p-nitroanilide hydrolysis reveals that the pK of the active site is significantly lowered (i.e. to approximately 6.3) in the enzyme-substrate complex. The temperature effect on the pK suggests the presence of a carboxylate in the active site of the enzyme. This suggestion is corroborated by the influence of organic solvent perturbations of the pK indicating a catalytic group of the neutral acid type. Values for the thermodynamic parameters associated with activation are reported. The transition state for ester degradation is at lower delta G and delta H levels than that for amide degradation. delta H and delta S for ester hydrolysis were noted to compensate each other with a compensation temperature Tc = 339K. Compensation can probably be related to weak bonding effects.