Determination of steady-state kinetic parameters for a xylanase-catalyzed hydrolysis of neutral underivatized xylooligosaccharides by mass spectrometry

被引:8
作者
Janis, Janne [1 ]
Pulkkinen, Petri [1 ]
Rouvinen, Juha [1 ]
Vainiotalo, Pirjo [1 ]
机构
[1] Univ Joensuu, Dept Chem, FI-80101 Joensuu, Finland
基金
芬兰科学院;
关键词
enzyme kinetics; substrate specificity; xylanase; xylooligosaccharide; electrospray ionization mass spectrometry;
D O I
10.1016/j.ab.2007.03.034
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A direct mass spectrometric approach was used for the determination of steady-state kinetic parameters, the turnover number (k(cat)), the Michaelis constant (Km), and the specificity constant (k(cat)/K-M) for an enzyme-catalyzed hydrolysis of xylooligosaccharides. Electrospray ionization mass spectrometry was performed to observe product distributions and to determine k(cat), K-M, and k(cat)/K-M values for Trichoderma reesei endo-1,4-beta-xylanase II (TRX II) with xylohexaose (Xyl(6)), xylopentaose (Xyl(5)), xylotetraose (Xyl(4)), and xylotriose (Xyl(3)) as substrates. The determined k(cat)/K-M values (0.93, 0.37, 0.027, and 0.00015 mu M-1 s(-1), respectively) indicated that Xyl(6) was the most preferred substrate of TRX II. In addition, the obtained K-M value for Xyl(5) (136 mu M) was roughly twice as high as that for Xyl(6) (73 mu M), suggesting that at least six putative subsites contribute to the substrate binding in the active site of TRX II. Previous mass spectrometric assays for enzyme kinetics have been used mostly in the case of reactions that result in a transfer of acidic groups (e.g., phosphate) into neutral oligosaccharides giving rise to negatively charged products. Here we demonstrate that such analysis is also feasible in the case of neutral underivatized oligosaccharides. Implications of the results for the catalytic mechanism of TRX II in particular are discussed. (c) 2007 Elsevier Inc. All rights reserved.
引用
收藏
页码:165 / 173
页数:9
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