Identification of Essential Ionizable Groups and Evaluation of Subsite Affinities in the Active Site of β-D-Glucosidase F₁ from a Streptomyces sp.

Abstract

  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 (A₁ was 1.10 kcal/mol and A₂ 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 (A₃ was −0.21 kcal/mol and A₄ was −2.8 kcal/mol).