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

• Published in: Journal of biochemistry (Tokyo) Vol. 127; no. 2; pp. 315 - 320
• Main Authors: Li, Y.K, Yao, H.J, Pan, I.H
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.02.2000; The Japanese Biochemical Society
• Subjects: analogs & derivatives; antagonists & inhibitors; aryl-beta-d-xylopyranosides; arylxylosides; Beta; Binding, Competitive; Bronsted plot; chemistry; Deuterium; Dithiothreitol; Dithiothreitol - pharmacology; enzyme activity; Enzyme Inhibitors; Enzyme Inhibitors - pharmacology; enzyme reaction mechanisms; enzymology; Gluconates; Gluconates - pharmacology; glycosides; Glycosides - metabolism; Hypocrea koningii; Kinetics; Lactones; Magnetic Resonance Spectroscopy; Mechanics; mechanism; metabolism; pharmacology; Pyrones; Pyrones - pharmacology; secondary deuterium isotope effect; Stereoisomerism; Substrate Specificity; substrates; Trichoderma; Trichoderma - enzymology; Trichoderma koningii; xylan; Xylose; Xylose - analogs & derivatives; Xylose - pharmacology; Xylosidases; Xylosidases - antagonists & inhibitors; Xylosidases - chemistry; Xylosidases - metabolism; β-xylosidase
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a022609

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-D-xylosides was found unequivocally to involve retention of the anomeric configuration. Based on the kCRt values of a series of arylxylosides with leaving group p.Kas 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-xylopyra-noside 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 (kHkD) 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