Transglucosidic reactions of the Aspergillus niger family 3 beta-glucosidase: qualitative and quantitative analyses and evidence that the transglucosidic rate is independent of pH.
The hydrolytic and transglucosidic reactions of the Aspergillus niger Family 3 beta-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 approximately 5.5 but increased at pH above approximately 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 beta-D-glucopyranosyl-(1-6)-beta-D-glucopyranosyl-p-nitrophenol and beta-D-glucopyranosyl-(1-6)-beta-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 approximately 5.5 but higher than Vmax(h) at pH greater than approximately 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 beta-linkages with oligosaccharide acceptors are important for acceptor reactivity.
Seidle
Heather F
HF
Division of Biochemistry, Faculty of Science, University of Calgary, Calgary, Alta., T2N1N4, Canada.
Huber
Reuben E
RE
D001234:Aspergillus niger / Q000201:enzymology*; D002475:Cellobiose / Q000737:chemistry; D002480:Cellulase / Q000737:chemistry; D002855:Chromatography, Thin Layer; D004305:Dose-Response Relationship, Drug; D005959:Glucosidases / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D016014:Linear Models; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D008956:Models, Chemical; D012044:Regression Analysis; D013696:Temperature; D013997:Time Factors; D001617:beta-Glucosidase / Q000737:chemistry*
D002475:Cellobiose; D005959:Glucosidases; D001617:beta-Glucosidase; D002480:Cellulase