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Abstract: Trp-262 of the Aspergillus niger family 3 β-glucosidase is shown in this report to be a key residue for determining the ratio of this enzyme’s hydrolytic and transglucosidic activities. TLC showed that when cellobiose was both the substrate and the acceptor, β-glucosidases with substitutions (Phe, Ala, Leu, and Cys) for Trp-262 formed very high amounts of transglucosidic adducts. When pNPGlc was the substrate and the acceptor of the substituted β-glucosidases, only transglucosidic adducts and pNP were produced. Little or no Glc could be detected, indicating that the reactions occurring were mainly transglucosidic. GLC studies with cellobiose quantitatively showed that one Glc was transferred for each free Glc produced. Since this is the maximum level of transglucosidation possible, this again showed that the reaction is predominantly transglucosidic. Analyses of the K m and K i values of cello-oligosaccharides of increasing length, of the K i values of Glc and of the transglucosidic activity at low acceptor concentration, showed that substitution for Trp-262 causes poor binding at the binding site for the non-reducing Glc of the substrate while the affinity for other Glc units is only minimally affected. The acceptor sites become saturated with substrate (acceptor) at the concentrations needed for glucosidic bond cleavage and thus only transglucosidic reactions occur. In addition, the data indicate that substitution for Trp-262 causes the rate of the hydrolysis step (k 3) to be small. [Copyright &y& Elsevier]

Abstract: The hydrolytic and transglucosidic reactions of the Aspergillus niger Family 3 β-glucosidase were characterized. Michaelis–Menten plots of the rates of aglycone formation were normal (hyperbolic) at low [substrate]. However, at high [substrate] the rates decreased at pH below ∼5.5 but increased at pH above ∼5.5. Each decrease or increase took the form of a second hyperbola adjoining the first. Thin layer chromatography, gas-liquid chromatography, and NMR analyses indicated that the substrates became transglucosidic acceptors when present at high concentrations. When pNPGlc and cellobiose reacted as acceptors, the C6 hydroxyl of the non-reducing substrate component reacted to form β-d-glucopyranosyl-(1–6)-β-d-glucopyranosyl-p-nitrophenol and β-d-glucopyranosyl-(1–6)-β-d-glucopyranosyl-(1–4)-d-glucopyranose, respectively. The acceptor action accounted for the second adjoining hyperbolas. Rate equations were derived for the production of the aglycone and the transglucosidic intermediate, and these equations described the data very well. Hydrolytic Vmax {Vmax(h)}, hydrolytic Km {Km(h)}, transglucosidic Vmax {Vmax(t)}, and transglucosidic Km {Km(t)} values were obtained by non-linear regression analysis using these equations. Vmax(h) pH profiles were bell shaped with optima between pH 4 and 4.5 but the Vmax(t) values did not change substantially between pH 3 and 7. These differences in the pH profiles explain the decreasing and increasing adjoining hyperbolas since Vmax(t) is lower than Vmax(h) at pH less than ∼5.5 but higher than Vmax(h) at pH greater than ∼5.5. The reason for these pH effects is that the value of the hydrolytic rate constant (k3) decreases while the value of the transglucosidic rate constant (k4) does not change between pH 3 and 7. The study also showed that gentiobiose forms by an intermolecular reaction of the C6 hydroxyl of Glc rather than an intramolecular reaction and that an equatorial orientation of the C2 hydroxyl, the presence of a C6 primary hydroxyl and β-linkages with oligosaccharide acceptors are important for acceptor reactivity. [Copyright &y& Elsevier]