STATIC AND KINETIC STUDIES BY FLUOROMETRY ON INTERACTION BETWEEN GLUCONOLACTONE AND GLUCOAMYLASE FROM RH-NIVEUS

Abstract

A transition-state analog, gluconolactone, was found to partially quench the protein fluorescence of glucoamylase [EC 3.2.1.3] from R. niveus. The interaction between gluconolactone and the enzyme was studied statically and kinetically at pH 4.5 in terms of fluorescence change. The Kd of the enzyme-analog complex determined by fluorometric titration at 25.degree. C (Kd = 1.6 mM) was in good agreement with that obtained by difference spectrophotometric titration and with the inhibitor constant determined for the hydrolysis of maltodextrin. The kinetics of the interaction were studied by the fluorescence stopped-flow method. The dependence of the apparent 1st-order rate constant, kapp, on gluconolactone concentration showed a saturation curve, consistent with a 2-step mechanism involving a rapid bimolecular association followed by a slow unimolecular isomerization process. The dissociation constant, KI, for the rapid bimolecular process and the forward and backward rate constants for the isomerization were obtained at 25 and 5.degree. C, and the activation parameters were evaluated. The isomerization process, but not the bimolecular association, is accompanied by fluorescence intensity change, indicating that the former process involves a microenvironmental change of a tryptophan residue(s) of the enzyme. Maltose decreased the rate of interaction of gluconolactone with the enzyme by competing with the analog for the active site.