Kinetic Studies on Substrate Specificity and Active Site of .BETA.-D-Glucosidase F1 from Streptomyces sp.

Abstract

The substrate specificity and the active site of β-D-glucosidase F-1 (Mr, 50, 000; optimum pH, 5.5) purified from a Streptomyces sp. were kinetically investigated. The β-D-glucosidase showed a broad substrate specificity for synthetic glycosides and disaccharides having β-glycosidic linkage, but the former was more favorable substrate than the latter. The enzyme was characterized by the ability to hydrolyze rapidly not only ρ-nitrophenyl β-glucoside (Km, 0.72 mM; k0, 63 s-1) but also ρ-nitrophenyl β-fucoside (Km, 0.19 mM; k0, 44 s-1) and laminaribiose (Km, 1.6 mM; k0, 70 s-1). The kinetic study was made as to whether the hydrolyses of these synthetic glycosides were catalyzed at a single active site or at dual active sites in the β-D-glucosidase. In the experiments with the mixed substrates of p-nitrophenyl β-glucoside (PNPG) and ρ-nitrophenyl β-fucoside (PNPF) or ρ-nitrophenyl β-galactoside (PNPGal), the kinetic features, the linearity of Lineweaver-Burk plots and the dependence of the apparent maximal veloities and Km value on the mole fraction (f) of PNPG in the mixed substrate, f = [PNPG/([PNPG] + [PNPF or PNPGal]) agreed very closely with those theoretically predicted for a single catalytic site mechanism. The findings strongly support the notion that the β-D-glucosidase attacks the synthetic glycosides at a common active site.