A steady-state kinetic analysis of the reaction between arginine esterase E-I from Bitis gabonica venom and synthetic arginine substrates and the influence of pH, temperature and solvent deuterium isotope.

1. Using synthetic arginines as substrates, steady-state kinetic studies showed a deviation from Michaelis-Menten kinetics for esterase E-I purified from the venom of Bitis gabonica. Graphical analysis indicated a rate equation of at least a degree of 3:3. 2. pH variation of the kinetic parameters indicated the involvement of groups with pK values of approximately 7 and approximately 9 which had to be in the ionic form for activity. 3. Solvent isotope studies suggested transition states where proton transfer or reorganization was the rate-limiting step of proteolytic catalysis. A single protogenic site was postulated. 4. Temperature effects on the enzymic reaction showed a significant reduction in entropy loss upon formation of the transition state with both esters and extended tail polypeptide-anilides in comparison with the activation entropy for benzoyl-L-arginine p-nitroanilide.

Inactivation of arginine esterase E-I of Bitis gabonica venom by irreversible inhibitors including a water-soluble carbodiimide, a chloromethyl ketone and isatoic anhydride.

1. Esterase E-I from Bitis gabonica was inactivated with irreversible inhibitors which included studies with a water-soluble carbodiimide, an affinity labelling peptide and a mechanism-based inactivator. 2. The reaction with 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide was biphasic and the dominant part followed saturation kinetics. At pH 5.5 a rate constant of 0.4 min-1 for inactive enzyme formation was calculated and a dissociation constant (Ki) of 0.2 M for the enzyme-inhibitor complex. 3. Inactivation with D-Phe-Pro-Arg-chloromethyl ketone indicated a two-step mechanism, for which the reaction parameters at pH 8.0 were determined. The Ki value was 0.2 microM and the inactivation rate was 2.5 min-1. 4. With isatoic anhydride pseudo-first-order kinetics was observed. At pH 8.0 a rate constant of 0.9 min-1 and a Ki of 2.0 mM were obtained. The inactivation of the enzyme was found to be governed by a group in the enzyme showing a pK value of 7.3.