Static and kinetic studies by fluorometry on the interaction between gluconolactone and glucoamylase from Rh. niveus.
A transition-state analog, gluconolactone, was found to partially quench the protein fluorescence of glucoamylase [EC 3.2.1.3] from Rhizopus niveus. The interaction between gluconolactone 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 fluorometric titration at 25 degrees (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 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 degrees and 5 degrees, and the activation parameters were evaluated. It was found that the isomerization process, but not the bimolecular association, is accompanied 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.
D001665:Binding Sites; D005942:Gluconates / Q000378:metabolism*; D005959:Glucosidases / Q000378:metabolism*; D007700:Kinetics; D007783:Lactones; D008320:Maltose / Q000494:pharmacology; D012233:Rhizopus / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
D005942:Gluconates; D007783:Lactones; D008320:Maltose; D005959:Glucosidases; C010730:beta-glucono-1,5-lactone