Mechanistic study of beta-xylosidase from Trichoderma koningii G-39

The catalytic mechanism of the beta-xylosidase purified from the culture fi ltrate of Trichoderma Koningii G-39 was investigated. By NMR spectroscopy, the stereochemistry of the enzyme catalyzing the hydrolysis of 2,4,dinitrop henyl and p-nitrophenyl-beta-D-xylosides was found unequivocally to involve retention of the anomeric configuration, Based on the k(cat) values of a s eries of arylxylosides with leaving group pK(a)s in the range of 4-10, an e xtended Bronsted plot was constructed with a slope (beta(Ig)) near zero. En zymatic hydrolysis of aryl-beta-D-xylosides in acetate buffer (pH 4.0) cont aining 3 or 5% methanol showed a constant product ratio (methylxyloside/xyl ose), indicating the presence of a common intermediate, probably the xylosy l-enzyme intermediate. In the presence of DTT, the k(cat) values of p-cyano phenyl-beta-D-xylopyranoside and p-nitrophenyl-beta-xylopyranoside increase d greatly. A two-step mechanism involving the formation and breakdown of th e xylosyl-enzyme intermediate was therefore proposed. The rate-limiting ste p is the breakdown of the intermediate. The secondary deuterium kinetic iso tope effect (k(H)/k(D)) measured for 2,4-dinitrophenyl-beta-D-xyloside was 1.02+/-0.01, suggesting that the transition state for breakdown of the xylo syl-enzyme intermediate is S(N)2-like.