Landt O, Thölke J, Grunert H P, Saenger W, Hahn U
Institut für Kristallographie, Freie Universität Berlin, Germany.
Biol Chem. 1997 Jun;378(6):553-8. doi: 10.1515/bchm.1997.378.6.553.
The enzymatic activity of many ribonucleases (RNases) depends on two histidines, as in RNase A, or one histidine and/or glutamate, as in microbial RNases belonging to the T1 family. In RNase T1, substitution of either one or both of the two histidines at positions 40 and 92 by a variety of other amino acids reduces the activity more than 100-fold. However, the double variant His40-->Asp/His92-->Asp retains a significant residual enzymatic activity towards RNA and guanylyl-3',5'-cytidine, indicating that a pair of substituted side chains in these positions, both with acid functionality, can confer enzymatic activity. It was shown that the substitution of histidine with glutamate in the variant His40-->Glu yields an enzyme with drastically reduced activity and leads to inactivation in the His92-->Glu, His40-->Glu/His92-->Glu variants. For the variants where histidine is substituted with aspartate we found measurable activity from 1% (His40-->Asp) to 6% (His40-->Glu/His92-->Asp) towards RNA.
许多核糖核酸酶（RNase）的酶活性取决于两个组氨酸，如在核糖核酸酶A中，或一个组氨酸和/或谷氨酸，如在属于T1家族的微生物核糖核酸酶中。在核糖核酸酶T1中，将第40位和第92位的两个组氨酸中的一个或两个用多种其他氨基酸取代，会使活性降低100倍以上。然而，双变体His40→Asp/His92→Asp对RNA和鸟苷-3',5'-胞苷仍保留显著的残余酶活性，这表明在这些位置上一对都具有酸性功能的取代侧链可赋予酶活性。结果表明，在变体His40→Glu中用谷氨酸取代组氨酸会产生一种活性大幅降低的酶，并导致His92→Glu、His40→Glu/His92→Glu变体失活。对于组氨酸被天冬氨酸取代的变体，我们发现其对RNA的活性可测量，从1%（His40→Asp）到6%（His40→Glu/His92→Asp）。
