Mechanistic study of beta-xylosidase from Trichoderma koningii G-39.
The catalytic mechanism of the beta-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-beta-D-xylosides was found unequivocally to involve retention of the anomeric configuration. Based on the k(cat) values of a series of arylxylosides with leaving group pK(a)s in the range of 4-10, an extended Bronsted plot was constructed with a slope (beta(lg)) near zero. Enzymatic hydrolysis of aryl-beta-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 k(cat) values of p-cyanophenyl-beta-D-xylopyranoside and p-nitrophenyl-beta-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 (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 xylosyl-enzyme intermediate is S(N)2-like.
Li
Y K
YK
Applied Chemistry Department, National Chiao Tung University, Hsin-Chu, Taiwan, 30050, Japan. ykl@cc.nctu.edu.tw
Yao
H J
HJ
Pan
I H
IH
D001667:Binding, Competitive; D003903:Deuterium; D004229:Dithiothreitol / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005942:Gluconates / Q000494:pharmacology; D006027:Glycosides / Q000378:metabolism; D007700:Kinetics; D007783:Lactones; D009682:Magnetic Resonance Spectroscopy; D019563:Mechanics; D011753:Pyrones / Q000494:pharmacology; D013237:Stereoisomerism; D013379:Substrate Specificity; D014242:Trichoderma / Q000201:enzymology*; D014994:Xylose / Q000031:analogs & derivatives / Q000494:pharmacology; D014995:Xylosidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*
D004791:Enzyme Inhibitors; D005942:Gluconates; D006027:Glycosides; D007783:Lactones; D011753:Pyrones; C408385:delta-xylonolactone; C010807:xylosides; D014994:Xylose; D003903:Deuterium; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase; D004229:Dithiothreitol; C010730:beta-glucono-1,5-lactone