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首页> 外文期刊>Biochemistry >EFFECTS OF BOTH SHORTENING AND LENGTHENING THE ACTIVE SITE NUCLEOPHILE OF BACILLUS CIRCULANS XYLANASE ON CATALYTIC ACTIVITY
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EFFECTS OF BOTH SHORTENING AND LENGTHENING THE ACTIVE SITE NUCLEOPHILE OF BACILLUS CIRCULANS XYLANASE ON CATALYTIC ACTIVITY

机译：延缓和增强芽孢杆菌木聚糖木聚糖酶活性位点核酸对催化活性的影响

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摘要

The relative positioning of the two carboxyl groups at the active site of glycosidases is crucial to their function and the mechanism followed. The distance between these two groups in Bacillus circulans xylanase has been modified by mutagenesis of the catalytic nucleophile Glu78. Ail increase in the separation (Glu78Asp) results in a large (1600-5000-fold) reduction in the rate of the glycosylation step, but little change In the extent of bond cleavage or proton donation at the transition state. A decrease in the separation tvas achieved by selective carboxymethylation of the Glu78Cys mutant, This modified mutant was only 16-100-fold less active than wild-type enzyme, and its transition state structure was similarly unchanged, Complete removal of the carboxyl group (Glu78Cys) resulted in a mutant with no measurable catalytic activity, Furthermore, it did not even undergo tile first step, glycosylation of the active site thiol, These results confirm the Importance of precise positioning of the nucleophile at the active site of these enzymes.
机译：两个羧基在糖苷酶活性位点的相对定位对它们的功能和遵循的机制至关重要。环形芽孢杆菌木聚糖酶中这两个基团之间的距离已通过诱变亲核试剂Glu78进行了修饰。所有分离（Glu78Asp）的增加都会导致糖基化步骤的速率大大降低（1600-5000倍），但过渡状态下键断裂或质子给体的程度变化很小。通过Glu78Cys突变体的选择性羧甲基化实现的分离tvas的降低，该修饰的突变体的活性仅比野生型酶低16-100倍，并且其过渡态结构类似地保持不变，完全去除了羧基（Glu78Cys ）导致没有可测量的催化活性的突变体，此外，它甚至没有经历第一步，即活性位点巯基的糖基化。这些结果证实了亲核试剂精确定位在这些酶的活性位点上的重要性。

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