Extended kinetic analysis of ribonuclease T1 variants leads to an improved scheme for the reaction mechanism.

Recombinant ribonuclease (RNase) T1 variants were characterized kinetically taking into account the different reactions catalyzed by this enzyme. In addition to established assays, monitoring the transesterification activity, a photometric assay for fast screening of RNase T1 and variants thereof for ester hydrolysis activity is described, which is based on the application of phenol red as pH indicator. Moreover we established an HPLC assay to evaluate RNase T1 variants by their ability to carry out the transesterification towards an internucleotide diphosphoester (reverse or synthetic activity). In this way we found that the transesterification and hydrolyzing activities of variants change in various directions though in all reactions the same active site and the same transition state are involved. The variant where Tyr42 has been replaced by Trp performs RNA synthesis better than the wild type protein. The scheme of the hypothetic RNase T1 mechanism had to be improved to take into account the non processive character of the reaction.