Substrate Specificity and Subsite Affinities of Honeybee α-Glucosidase II

Abstract

The substrate specificity of honeybee α-glucosidase II, a monomeric protein, was investigated in de-tail. The enzyme hydrolyzed phenyl α-glucoside and p-nitrophenyl α-glucoside more rapidly than maltooligosaccharides. Unusual kinetics were observed in hydrolysis of sucrose, turanose, kojibiose, and soluble starch. The s versus v plots showed sigmoidal curves, and so the Lineweaver-Burk plots became concave, indicating positive kinetic cooperativity. The Hill coefficients for sucrose, turanose, kojibiose, and soluble starch were calculated to be 1.46 ; 1.34, 1.15, and 1.28, respectively. The K_m values for these substrates were calculated as the concentration at one half of the maximum velocity. The ratios of the maximum velocities for maltose, malto-triose, -tetraose, -pentaose, -hexaose, -heptaose, maltodextrin (DP=13), kojibiose, nigerose, isomaltose, sucrose, turanose, phenyl α-glucoside, p-nitrophenyl α-glucoside, phenyl α-maltoside, and soluble starch were estimated to be 100 : 163 : 159 : 156 : 149 : 144 : 113 : 96.9 : 92.2 : 31.8 : 81.5 : 68.8 : 268 : 341 : 127 : 17.0, and the K_m values for these substrates, 5.4, 4.0, 6.3, 11, 31, 50, 50, 7.6, 20, 5.6, 6.7, 7.7, 1.6, 1.8, 2.0, and 9.4 mM, respectively. The enzyme was also active on α-glucose-1-phosphate. Its maximum activity was 79.9% of that to maltose and the K_m was 50mM. The substrate specificity of this enzyme was different from that of honeybee α-glucosidase I. The two enzymes were found to be immunologically distinct proteins. Based on the rate parameters for maltooligosaccharides, the subsite affinities (A_i's) in the active site of the enzyme were evaluated. Subsites 1, 2, and 3 having the positive A_i value (A₁, A₂, and A₃ : 0.672, 4.61, and 0.483kcal/mol, respectively) were considered to be effective for binding of substrate to the active site.