Abstract
The catalytic mechanism of the β-xylosidase purified from the culture filtrate of Trichoderma koningii G-39 was investigated. By NMR spectroscopy, the stereochemistry of the enzyme catalyzing the hydrolysis of 2, 4-dinitrophenyl and p-nitrophenyl-β-xylosides was found unequivocally to involve retention of the anomeric configuration. Based on the kcat values of a series of arylxylosides with leaving group pKas in the range of 4-10, an extended Bronsted plot was constructed with a slope (β1g) near zero. Enzymatic hydrolysis of aryl-β-D-xylosides in acetate buffer (pH 4.0) containing 3 or 5% methanol showed a constant product ratio (methylxyloside/xylose), indicating the presence of a common intermediate, probably the xylosyl-enzyme intermediate. In the presence of DTT, the kcat values of p-cyanophenyl-β-D-xylopyranoside and p-nitrophenyl-β-D-xylopyranoside increased greatly. A two-step mechanism involving the formation and breakdown of the xylosyl-enzyme intermediate was therefore proposed. The rate-limiting step is the breakdown of the intermediate. The secondary deuterium kinetic isotope effect (kH/kD) measured for 2, 4-dinitrophenyl-β-D-xyloside was 1.02±0.01, suggesting that the transition state for breakdown of the xylosyl-enzyme intermediate is SN2-like.
