Multiple substrate monitoring

Information concerning enzyme specificity can be obtained by monitoring the reaction with several different substrates simultaneously. The time intensity curves for the respective substrates and products provide information on preferences of the enzyme used and on possible alterations of the mechanism in enzyme variants. For RNase T1, multiple substrate monitoring has been used to compare the properties of the RV variant with RNase T1 wild type. As RNase T1 RV is less specific for guanosine in the $G_S^B$ position than RNase T1 wild type, the simultaneous cleavage of 5',3'-GpC and 5',3'-ApC was monitored. Using RNase T1 wild type, no hydrolysis of 5',3'-ApC could be detected. In contrast to this, on-line monitoring of the enzymatic hydrolysis of the dinucleoside monophosphate mixture by RNase T1 RV exhibited an enzymatic transesterification of 5',3'-ApC too (see figure 3.13), even though the reaction rate is very low due to the reduced activity of the variant.

To increase the reaction rate and to ascertain, wether RNase T1 RV follows an altered reaction mechanism with an accelerated hydrolysis of 2',3'-cAMP - as indicated by TLC-investigations (RIKO CZAJA, personal communication) - a ten-fold increased enzyme concentration (200 nM) has been used (see figure 3.14). Under these conditions, the equilibrium state of the RNase T1 RV catalysed transesterification of 5',3'-GpC to 2',3'-cGMP and cytidine (data for cytidine not shown) - with almost no detectable change in intensity of the signal for the substrate - could be observed within thirty minutes, whereas the signal for the intensity of 5',3'-ApC did not show unequivocal signs of constance. Expectedly, the intensity of the 2',3'-cGMP signal increased during the observation time. However, the intensity curve for the final product of 5',3'-ApC hydrolysis, 3'-AMP, remained almost unchanged during the complete observation time. This reveals, that RNase T1 RV does not follow an altered reaction mechanism with an increased cyclic phosphodiester hydrolysis.

Figure 3.13: On-line monitoring of multiple substrate cleavage (5',3'-ApC and 5',3'-GpC) by RNase T1 RV.
Conditions: 750 $\mu$M 5',3'-GpC and 5',3'-ApC in 10 mM ammonium acetate solution (pH 6.5), room temperature, RNase T1: 20 nM

Figure 3.14: Multiple substrate on-line monitoring of the hydrolysis of 5',3'-ApC and 5',3'-GpC by RNase T1 RV with elevated enzyme concentration.
Conditions: 750 $\mu$M 5',3'-GpC and 5',3'-ApC in 10 mM ammonium acetate solution (pH 6.5), room temperature, RNase T1 200 nM