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. Biochein. 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, k.pp, 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, Kr, for the rapid bimolecular process and the forward and backward rate constants for the isomerization were obtained at 25° and Y, and the activation parameters were evaluated.