Reverse Action of Ribonuclease T1 Variants In ICE

Modification of Ribonuclease T1 Specificity by Random Mutagenesis of the Substrate Binding Segment,

“…Further studies on substrate specificity were carried out by using the reverse transesterification reaction of the enzymes for synthesis experiments. Recently, we established that RNase T1-catalyzed GpC synthesis can take advantage of the yield-increasing effect of freezing the reaction mixtures (35), as already reported for proteasecatalyzed peptide synthesis (39). In this work, we observed a strong influence of the wt enzyme concentration on GpC yield (35).…”

“…Recently, we established that RNase T1-catalyzed GpC synthesis can take advantage of the yield-increasing effect of freezing the reaction mixtures (35), as already reported for proteasecatalyzed peptide synthesis (39). In this work, we observed a strong influence of the wt enzyme concentration on GpC yield (35). Therefore, we studied the synthesis of ApC and GpC starting from the 2′3′-cyclic phosphodiesters and cytidine at various concentrations of RNase T1-8/3 in solution at 0 °C as well as in frozen reaction systems at -10 °C.…”

“…The influence of the enzyme concentrations on the dinucleoside monophosphate yield was more distinct for GpC than for ApC synthesis. Furthermore, we compared the synthesis potentials of RNase T1-8/3 and wt enzyme using for both enzymes concentrations which have been proven to be optimal in ApC synthesis catalyzed by RNase T1-8/3, and in wt-catalyzed GpC synthesis (35). No remarkable differences between wt and variant could be observed concerning GpC synthesis, whereas ApC synthesis catalyzed by RNase T1-8/3 showed a 7-fold increased yield compared to the wt enzyme (Figure 2C,D).…”

“…HPLC analysis was performed as described earlier (35). In the case of ApC synthesis and cleavage, the components were separated by gradient elution with 0.01 M potassium dihydrogen phosphate (pH 4.15) and 0-30% methanol within 10 min followed by an increase in methanol concentration up to 50% within a further 5 min.…”

“…HPLC Analysis. HPLC analysis was performed as described earlier (35) 0-30% methanol within 10 min followed by an increase in methanol concentration up to 50% within a further 5 min. 3′AMP, 2′3′cAMP, and ApC were detected at 268 nm.…”