The Proflavin-Induced Increase in the Catalytic Activity of Ficin.

The dye proflavin (3,6 diaminoacridine) binds to a single site on the ficin molecule (EC 3.4.4.12). The value of the dissociation constant (Kp) 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 (kcat) for the ficin-catalysed hydrolysis of benzoyl-L-arginine ethyl ester although the value of KM app was unaltered. Dye-binding did not significantly affect the value of kcat 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 Kp=1.61 7times; 10-4 M assuming a kinetic model involving formation of a ternary dye-enzyme-substrate complex in which substrate is more rapidly hydrolyzed 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-arginine ethyl ester hydrolysis by ficin are discussed, including consideration of an allosteric modification mechanism. [ABSTRACT FROM AUTHOR]