Identification of Essential lonizable Groups and Evaluation of Subsite Affinities in the Active Site of β-D-Glucosidase F_1 from a Streptomyces sp.(Biochemistry & Molecular Biology)

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Partial amino acid sequences, the essential ionizable groups directly involved in catalytic reaction, and the subsite structure of β-D-glucosidase purified from a Streptomyces sp. were investigated in order to analyze the reaction mechanism. On the basis of the partial amino acid sequences, the enzyme seemed to belong to the family 1 of β-glucosidase in the classification of glycosyl hydrolases by Henrissat (1991). Dependence of the V and K_m values on pH, when the substrate concentration was sufficiently lower than K_m, gave the values of 4.1 and 7.2 for the ionization constants, pK_<e1> and pK_<e2> of essential ionizable groups 1 and 2 of the free enzyme, respectively. When the dielectric constant of the reaction mixture was decreased in the presence of 10% methanol, the pK_<e1> and pK_<e2>, values shifted to higher, to +0.60 and +0.35 pH unit, respectively. The findings supported the notion that the essential ionizable groups of the enzyme were a carboxylate group (-COO^-, the group 1) and a carboxyl group (-COOH, the group 2). The subsite affinities A_i's in the active site were evaluated on the basis of the rate parameters of laminarioligosaccharides. Subsites 1 and 2 having positive A_i values (A_1 was 1.10 kcal/mol and A_2 was 4.98 kcal/mol) were considered to probably facilitate the binding of the substrate to the active site. However, the subsites 3 and 4 showed negative A_i values (A_3 was -0.21 kcal/mol and A_4 was -2.8 kcal/mol).
社団法人日本農芸化学会の論文
2002-10-23

Identification of Essential lonizable Groups and Evaluation of Subsite Affinities in the Active Site of β-D-Glucosidase F_1 from a Streptomyces sp.(Biochemistry & Molecular Biology)

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