Open AccessThe Proflavin‐Induced Increase in the Catalytic Activity of Ficin
Author(s) -
Hollaway M. R.
Publication year - 1968
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1968.tb00379.x
Subject(s) - chemistry , hydrolysis , ternary complex , catalysis , acylation , nucleophile , substrate (aquarium) , medicinal chemistry , reaction rate constant , stereochemistry , enzyme , organic chemistry , kinetics , oceanography , geology , physics , quantum mechanics
1 The dye proflavin (3,6 diaminocaridine) binds to a single site on the ficin molecule (EC 3.4.4.12). 2 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. 3 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. 4 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. 5 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. 6 Dye‐binding leads to an enhanced nucleophilicity of the essential thiol group of ficin. 7 It is concluded that deacylation is not the rate‐limiting step in the ficin‐catalysed hydrolysis of benzoyl‐L‐arginine ethyl ester. 8 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.