EXTENDED KINETIC-ANALYSIS OF RIBONUCLEASE-T1 VARIANTS LEADS TO AN IMPROVED SCHEME FOR THE REACTION-MECHANISM

Recombinant ribonuclease (RNase) T1 variants were characterized kineti cally taking into account the different reactions catalized by this en zyme. In addition to established assays, monitoring the transesterific ation 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 e stablished an HPLC assay to evaluate RNase T1 variants by their abilit y to carry out the transesterification towards an internucleotide diph osphoester (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 b een replaced by Trp performes RNA synthesis better than the wild type protein. The scheme of the hypothetic RNase T1 mechanism had to be imp roved to take into account the non processive character of the reactio n. (C) 1994 Academic Press, Inc.