Lawson S L, Wakarchuk W W, Withers S G
Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, Canada.
Biochemistry. 1996 Aug 6;35(31):10110-8. doi: 10.1021/bi960586v.
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. An 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 was 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 the 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.
糖苷酶活性位点上两个羧基的相对位置对其功能及作用机制至关重要。通过对催化亲核试剂Glu78进行诱变，已改变了环状芽孢杆菌木聚糖酶中这两个基团之间的距离。间距增加（Glu78Asp）会导致糖基化步骤的速率大幅降低（1600 - 5000倍），但在过渡态时键断裂程度或质子供体情况变化不大。通过对Glu78Cys突变体进行选择性羧甲基化实现了间距减小。这种修饰后的突变体活性仅比野生型酶低16 - 100倍，并且其过渡态结构同样未改变。完全去除羧基（Glu78Cys）会产生一个没有可测量催化活性的突变体。此外，它甚至不会经历第一步，即活性位点硫醇的糖基化。这些结果证实了亲核试剂在这些酶活性位点上精确定位的重要性。
