期刊
EUROPEAN JOURNAL OF BIOCHEMISTRY
卷 271, 期 21, 页码 4169-4177出版社
WILEY-BLACKWELL
DOI: 10.1111/j.1432-1033.2004.04354.x
关键词
beta-glycosidase; bioenergetics analysis; enzyme specificity; glycoside hydrolase; site-directed mutagenesis
The specificity of the Spodoptera frugiperda digestive beta-glycosidase (Sfbetagly50) for fucosides, glucosides and galactosides is determined by noncovalent interactions of glycone 6-OH and glycone 4-OH with the active-site residues Q39 and E451. Site-directed mutagenesis and enzyme steady-state kinetics were described, showing that replacement of E451 with glutamine increased the preference of Sfbetagly50 for glucosides in comparison to galactosides, whereas replacing E451 with serine had the opposite effect. In contrast, the replacement of E451 with aspartate did not change Sfbetagly50 specificity. The energy of the interactions formed by these different residues with the axial and equatorial glycone 4-OH were also measured, showing that the increase in preference for galactosides resulted from a larger energy decrease in the interaction with equatorial 4-OH than with axial 4-OH (22.6 vs. 13.9 kJ.mol(-1)), whereas the increase in preference for glucosides was caused by an energy reduction in the interaction with the axial 4-OH (5.1 kJ.mol(-1)). The introduction of glutamine at position 451 or of asparagine at position 39 increased the preference of Sfbetagly50 for fucosides in comparison to galactosides, whereas the presence of aspartate or serine at position 451 had less effect on this preference. The hydrolysis of fucosides was favored because glutamine at position 451 increased a steric hindrance with 6-OH of 7.1 kJ.mol(-1) and asparagine at position 39 disrupted a favorable interaction with this same hydroxyl. In conclusion, it is proposed that the specificity of new beta-glycosidase mutants can be predicted by combining and adding energy of the enzyme-substrate interactions evaluated in the present study.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据