5',3'-ApC transesterification

The acquisition of kinetic parameters for 5',3'-ApC transesterification to 2',3'-cAMP and cytidine is difficult to monitor spectrophotometrically due to the high self-absorption of the substrate. Therefore it is difficult to determine kinetic parameters of RNase T1 variants which have their specificity altered towards adenosine at their $G_S^B$ binding site regarding the catalysed turnover of 5',3'-ApC. RNase T1 RV has a modified substrate specificity (see 1.2.2) and is able to hydrolyse a substrate with adenosine at the $G_S^B$ position. Mass spectrometric off-line reaction monitoring was used to determine kinetic parameters for the turnover of 5',3'-ApC catalysed by RNase T1 RV. Substrate concentrations between 50 $\mu$M and 2,500 $\mu$M and an enzyme concentration of 50 nM were used in the assay conducted, the reaction was monitored under steady-state conditions. The parameters determined are shown in table 3.2, the MICHAELIS-MENTEN curve (substrate concentration-rate curve) can be seen in figure 3.5. In contrast to RNase T1 wild type, where a large amount of kinetic data is available for the 5',3'-GpC transesterification, there are no published data concerning the 5',3'-ApC transesterification by RNase T1 RV. For this reason, it is yet impossible to compare the parameters obtained using mass-spectrometric off-line reaction monitoring.

Table 3.2: Kinetic parameters for 5',3'-ApC transesterification by RNase T1 RV acquired by off-line reaction monitoring.
Conditions: 10mM ammonium acetate (pH 6.5), 5',3'-ApC: 50 to 2,500 $\mu$M, RNase T1 RV 50nM, room temperature, n=12

K$_m$	$\mu$M	1,174	$\pm$ 35%
k$_{cat}$	s$^{-1}$	89	$\pm$ 17%

Figure 3.5: Concentration-rate data for 5',3'-ApC transesterification by RNase T1 RV, obtained using off-line reaction monitoring. The figure shows the MICHAELIS-MENTEN-curve with the parameters estimated from the data acquired ($K_m$ = 1,174 $\mu$M, $k_{cat}$ = 89 $s^{-1}$).
Conditions: 10mM ammonium acetate (pH 6.5), 5',3'-ApC: 50 to 2,500 $\mu$M, RNase T1 RV 50nM, room temperature, n=12