J. Biochem, 1990, Vol. 107, No. 5 762-768
© 1990 Japanese Biochemical Society
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Allosteric Properties, Substrate Specificity, and Subsite Affinities of Honeybee 
-Glucosidase I
Department of Agricultural Chemistry, Faculty of Agriculture, Hokkaido University Kita-ku, Sapporao, Hokkaido 060
1To whom correspondence should be addressed.
The substrate specicfieity of Honeybee 
-glucosidase I, a monomeric enzyme, was kinetically investighated. Unusual kinetic features were observed in the cleacage reactions of sucrose, maltose, p-nitropheny -glucoside, phenyl -gluscoside, turanose, and maltodextrin (DP= 13). At relatively high substrate concentrations, the velocities of liberation of fructose from sucrose, glucose from maltose, p-nitrophenol from p-nitrophenyl a-glucoside, and phenol from phenyl a-glucoside were accelerate, and so the Linewear-Burk plots were conver, indicating negative kinetic cooperativity: the Hill coefficients were calculated to be 0.50, 0.64, and 0.67 for sucrose, maltose, p-nitrophenylglucoside, and phenyl -glucoside, respectively. For the degradation of turanose and maltodextrin, the enzyme showed a sigmoidal curve in v versus s plots and thus catalyzed the reaction with positive kinetic cooperativity. The Lineweaver-Bruk plots were concave and the Hill coefficients were 1, 2 and 1.5 for turnanse and malthdextrin, respectively. These unique properties cannot be interpreted by the reaction mechanicm that Huber and Thompson proposed: (1973) Biochemistry 12, 4011–4020. The rate parameters for the bydrolysis of sucrose, maltose, p-nitrophenyl glucoside and pheny; glucoside were estimated by extrapolsting the linear part of the Lineweaver-Burk plots at low substrate concentrations.The ratios of the Vmex values for maltose, sucrose, kojibiose, p-nitrophenyl glucoside, phenyl pentaose, -hexasos, -heptaose and -octase, were estimated to be 100: 95: 81: 126: 85: 86: 113: 117: 111: 104: 95: 95, and the k values for these substates were 0.85, 4.2, 25, 0.31, 0.60, 0.41, 0.62, 2.0, 8.0, 15, 30, and 33 mM, respectively. Based on the rate parameters for maltooligosacchrides, the subsite affinities (A, s) in the active site of the enzyme were evausted. Subsite 1, 2and 3 having positive At values (A1, A2, and A3: 1.34, 5.37, and 0.269 kcal/mol, respectively) were considered to be effective for the binding of substrate to the active site.
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