Using point mutated overproducing strains of E.coli , ribonuclease T1 was prepared with the single substitutions Tyr24Trp, Tyr42Trp, Tyr45Trp or Trp59Tyr and the corresponding double substitutions Tyr24Trp/Trp59Tyr, Tyr42Trp/Trp59Tyr and Tyr45Trp/Trp59Tyr. Steady state kinetics of the transesterification reaction for the two dinucleoside monophosphate substrates guanylyl-3′, 5′-cytidine and guanylyl-3′, 5′-adenosine indicate that the tryptophan can be introduced in different positions within the ribonuclease T1 molecule without abolishing enzymatic activity. The Trp59Tyr exchange even enhances catalysis of the cleavage reaction ( 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 reaction of wild type ribonuclease T1 and of the variants, typically bell-shaped curves are observed with a plateau in the range pH 4.5–7.0. Their shapes and slopes indicate that the enzymes are comparable in their macroscopic p K a , values. At pH 7.5, the variant Tyr45Trp/Trp59Tyr shows a more than 3-fold higher transesterification activity for guanylyl-3′, 5′-adenosine and a 2-fold increase for guanylyl-3′, 5′-cytidine compared to the wild type enzyme, i.e. this variant catalyses the transesterification of the substrate guanylyl-3′, 5′-adenosine with the same or better efficiency as guanylyl-3′, 5′-cytidine.
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