TRP59 TO TYR SUBSTITUTION ENHANCES THE CATALYTIC ACTIVITY OF RNASE T1AND OF THE TYR TO TRP VARIANTS IN POSITION-24, POSITION-42 AND POSITION-45

Using point mutated overproducing strains of E.coli, ribonuclease T1 w as prepared with the single substitutions Tyr24Trp, Tyr42Trp, Tyr45Trp or Trp59Tyr and the corresponding double substitutions Tyr24Trp/Trp59 Tyr, Tyr42Trp/Trp59Tyr and Tyr45Trp/Trp59Tyr. Steady state kinetics of the transesterification reaction for the two dinucleoside monophospha te substrates guanylyl-3',5'-cytidine and guanylyl-3',5'-adenosine ind icate that the tryptophan can be introduced in different positions wit hin the ribonuclease T1 molecule without abolishing enzymatic activity . The Trp59Tyr exchange even enhances catalysis of the cleavage reacti on (k(cat)/K(m)) relative to the wild type enzyme and similar effects are found with single tyrosine to tryptophan substitutions. For the pH dependencies of the guanylyl-3',5'-cytidine transesterification react ion of wild type ribonuclease T1 and of the variants, typically bell-s haped curves are observed with a plateau in the range pH 4.5-7.0. Thei r shapes and slopes indicate that the enzymes are comparable in their macroscopic pK(a) values. At pH 7.5, the variant Tyr45Trp/Trp59Tyr sho ws a more than 3-fold higher transesterification activity for guanylyl -3',5'-adenosine and a 2-fold increase for guanylyl-3',5'-cytidine com pared to the wild type enzyme, i.e. this variant catalyses the transes terification of the substrate guanylyl-3',5'-adenosine with the same o r better efficiency as guanylyl-1-3',5'-cytidine.