Kinetic studies on coenzyme binding and coenzyme dissociation in tryptophanase immobilized on sepharose.

Abstract

The binding rate of pyridoxal '5-phosphate (Pxa-P) to apotryptophanase and the dissociation rate of the coenzyme from holotryptophanase were able to be determined by following the enzyme activity in continuous flow reactions on a column of immobilized tryptophanase. When the enzyme activity was assayed continuously in the flow system in the absence of coenzyme added to the reaction mixture, immobilized holotryptophanase lost gradually its initial activity owing to dissociation of coenzyme. The coenzyme dissociation at a given concentration of substrate (tryptophan) followed first-order kineticsmin a low substrate concentration range below the Km value, a more decreased rate constant was obtained for the coenzyme dissociation. This indicates that the coenzyme is more dissociable from the apoenzyme-coenzyme-substrate complex (ECS complex) rather than from the apoenzyme-coenzymecomplex (holoenzyme). Immobilized tryptophanase freed of coenzyme restored rapidly its original activity, when the assay mixture containing a given concentration of substrate and Pxa-P was passed through the immobilized enzyme column. The coenzyme binding at a given coenzyme concentration followed first-order kinetics, but the rate was not first order in regard to the coenzyme concentration. A plot of the reciprocal of the first-order rate constant obtained vs. the reciprocal of the coenzyme binding occurs in a two-step fashion; the first step is rapid and the second step is rate determining. Both the dissociation constant for the first step and the rate constant for the second step were shown to be independent of the substrate concentration. This means that Schiff base formation between Pxa-P and tryptophan in the assay mixture has no effect on the binding of Pxa-P to apoenzyme. The coenzyme dissociation constant at a given substrate concentration was calculated from both the rate constant of the coenzyme binding and the rate constant of the coenzyme dissociation. The values obtained by this method at different substrate concentrations were almost identical with those measured at the corresponding substrate concentrations directly by an ordinary method.