Papain action at neutral and alkaline pH regions

A new method for the determination of individual pre-steady-state parameters describing papain action is reported. This procedure has been developed from the quantitative analysis of steady-state kinetics concerning the enzymatic hydrolysis of a specific chromogenic substrate, namely N-α-carbobenzoxy-L-valine p-nitrophenyl ester. The analysis of steady-state kinetics allows: (i) determination of values of Ks (the fast dissociation pre-equilibrium constant for the formation of the reversible initial enzyme-substrate complex) and k+2 (the acylation rate constant) also under experimental conditions (.e., pH > 6.0) where usual methods, based on pre-steady-state measurements, do not hold; and (ii) experimental determination of values of k+3 (the deacylation rate constant) commonly calculated from values of catalytic parameters obtained separately under steady-state and pre-steady-state conditions. This procedure allowed experimental determination of both overall steady-state parameters and individual pre-steady-state quantities for papain catalysis, not only at acid pH values but also over neutral and alkaline pH regions (i.e., between pH 2.5 and 10.5). The effect of pH on papain action is analyzed in detail, and its possible general significance for cysteine proteinase-catalyzed reactions is discussed.