Static and Kinetic Studies by Fluorometry on the Interaction between Gluconolactone and Glucoamylase from Rh. niveus
1977 Volume 81 Issue 1 Pages 99-105
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1977 Volume 81 Issue 1 Pages 99-105
A transition-state analog, gluconolactone, was found to partially quench the protein fluores-cence of glucoamylase [EC 3. 2. 1. 3] from Rhizopus niveus. The interaction between glucono-lactone and the enzyme was studied statically and kinetically at pH 4.5 in terms of fluorescence change. The dissociation constant Kd of the enzyme-analog complex determined by fluoro-metric titration at 25° (Kd=1.6 mM) was in good agreement with that obtained by difference spectrophotometric titration (Ohnishi, M. et al. (1975) J. Biochem. 77, 695-703) and with the inhibitor constant determined for the hydrolysis of maltodextrin (Ohnishi, M. et al. (1976) J. Biochem. 79, 1007-1012).
The kinetics of the interaction were studied by the fluorescence stopped-flow method. The dependence of the apparent first-order rate constant, kapp, on gluconolactone concentration showed a saturation curve, consistent with a two-step mechanism involving a rapid bimolec-ular 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°, and the activation parameters were evaluated.
It was found that the isomerization process, but not the bimolecular association, is accom-panied by fluorescence intensity change, indicating that the former process involves a micro-environmental change of a tryptophan residue(s) of the enzyme.
Maltose was found to decrease the rate of interaction of gluconolactone with the enzyme by competing with the analog for the active site.
