Deviation from Michaelis - Menten kinetics for esterase E-II from the venom of Bitis gabonica, using synthetic arginine amides as substrates

Abstract

Steady state kinetic studies on the reaction between esterase E-II from the venom of Bitis gabonica and the fluorogenic substrates, N-α-benzoyl- l-phenylalanyl- l-valyl- l-arginine-4-methylcoumaryl-7 and N-α-benzoyl- l-arginine-4-methylcoumaryl-7-amide were found to deviate from Michaelis-Menten kinetics. Analysis of algebraic graphs and application of non-linear regression allowed an empirical rate law to be selected. The results revealed a rate equation of at least third degree at pH values above 7.0 and of 2:2 degree in the pH range 6–7. The data were interpreted in terms of a molecular model involving an enzyme with one catalytic site and several auxiliary or regulatory sites which, through cooperative effects, may either activate or inhibit the enzyme. Substrate activation is observed at low substrate values and might follow from an obligatory order of binding involving two of the sites, the modifier substrate molecule binding before the substrate molecule undergoing transformation to products. Inhibitory sites apparently become available only at alkaline pH. The inhibition is only noted at high substrate concentrations and is of the partial type.