The Proflavin‐Induced Increase in the Catalytic Activity of Ficin

Abstract

The dye proflavin (3,6 diaminocaridine) binds to a single site on the ficin molecule (EC 3.4.4.12). The value of the dissociation constant ( K p ) for the enzyme‐dye complex was 3.5 ± 1.8 × 10 −4 M measured spectrophotometrically and 1.55 ± 0.61 × 10 −4 by equilibrium dialysis. Interaction with proflavin increased the catalytic constant ( k cat ) for the ficin‐catalysed hydrolysis of benzoyl‐L‐arginine ethyl ester although the value of K M app was unaltered. Dye‐binding did not significantly affect the value of k cat for the ficin‐catalysed hydrolysis of p ‐nitrophenyl hippurate, a substrate for which acylation is not rate‐limiting in catalysis. The dependence of initial velocity of benzoyl‐ l ‐arginine ethyl ester hydrolysis on dye concentration enabled the computation of a value of K p = 1.61 × 10 −4 M assuming a kinetic model involving formation of a ternary dye‐enzyme‐substrate complex in which substrate is more rapidly hydrolysed than in the binary enzyme‐substrate complex. Dye‐binding leads to an enhanced nucleophilicity of the essential thiol group of ficin. It is concluded that deacylation is not the rate‐limiting step in the ficin‐catalysed hydrolysis of benzoyl‐L‐arginine ethyl ester. Possible explanations of the proflavin‐induced acceleration of benzoyl L‐arginie ethyl ester hydrolysis by ficin are discussed, including consideration of an allosteric modification mechanism.