On the Properties of alpha-Glucosidase and the Binding of Glucose to the Enzyme.

Abstract

An .alpha.-glucosidase (EC 3.2.1.20) was purified from baker''s yeast. The MW was approximately 44,000 daltons. SDS-disc gel electrophoresis suggested that the enzyme consisted of 4 subunits. The isoelectric point was at pH 5.4. The Km vaules for p-nitrophenyl .alpha.-D-glucopyranoside and maltose were 2.9 .times. 10-4 and 2.5 .times. 10-2 M, respectively. Binding of 2-(p-toluidino)naphthalene-6-sulfonate [TNS] to the .alpha.-glucosidase was associated with a strong increase in fluorescence. The dissociation constant of the enzyme-TNS complex was 8 .times. 10-5 M. The fluorescent probe did not interfere with the binding of glucose to the enzyme although the .alpha.-glucosidase was inhibited by high concentrations of TNS. The formation of an enzyme-glucose complex was indicated by an increase of fluorescence and by a shift in the wavelength for maximal emission which suggests that the binding process is associated with a change in conformation. The dissociation constant of the glucose-.alpha.-glucosidase complex KD = 0.57 .times. 10-3 M, was calculated from the increase in fluorescence as a function of glucose concentration.