5',3'-GpC transesterification

The standard assay for the determination of RNase T1 reaction parameters is the acquisition and analysis of initial rate curves for the transesterification of 5',3'-GpC to 2',3'-cGMP and cytidine acquired under steady-state conditions. For the assay conducted, substrate concentrations between 50 $\mu$M and 2,500 $\mu$M and an enzyme concentration of 5 nM were chosen. A typical initial rate curve acquired using off-line reaction monitoring is presented in figure 3.3. There is a linear correlation between the reaction time and 5',3'-GpC concentration, indicating, that the reaction was monitored under steady-state conditions. A curve showing the reaction rates at different 5',3'-GpC concentrations acquired using RNase T1 wild type is shown in figure 3.4. A variance of the rates determined can be seen. This is due to the difficulties in the reproducible manual handling small volumes below one microlitre, apart from the normal error in the acquisition of enzyme kinetic data. Using an autosampler device with sample preparation capabilities would increase the reproducibility of the data acquired and thus reduce the variance.

Figure 3.3: Time-concentration data for 5',3'-GpC transesterification by RNase T1 wild type acquired using off-line reaction monitoring.
Conditions: 10mM ammonium acetate (pH 6.5), 5',3'-GpC: 200$\mu$M, RNase T1 5nM, room temperature

Table 3.1: Kinetic parameters for 5',3'-GpC transesterification by RNase T1 wild type obtained by off-line reaction monitoring.
Conditions: 10mM ammonium acetate (pH 6.5), 5',3'-GpC: 50 to 2,500 $\mu$M, RNase T1 5nM, room temperature, n=13

K$_m$	[$\mu$M]	580	$\pm$	19%
k$_{cat}$	[s$^{-1}$]	205	$\pm$	7%

Figure 3.4: Concentration-rate data for 5',3'-GpC transesterification by RNase T1 wild type, acquired with off-line reaction monitoring. The figure shows the MICHAELIS-MENTEN-curve with the parameters estimated from the data obtained ($K_m$ = 580 $\mu$M, $k_{cat}$ = 205 $s^{-1}$).
Conditions: 10mM ammonium acetate (pH 6.5), 5',3'-GpC: 50 to 2,500 $\mu$M, RNase T1 5nM, room temperature, n=13

The kinetic parameters acquired for this reaction for RNase T1 wild type are shown in table 3.1. As the observation of the transesterification of 5',3'-GpC to form 2',3'-cGMP and cytidine is the standard assay for the investigation of RNase T1, a large amount of kinetic parameters for this reaction is available. However, only few data presented in literature are comparable with the data acquired herein as there are no standard conditions for the acquisition of kinetic parameters. In general, the data published show higher k$_{cat}$ and lower K$_m$ values. However, compared with the assays described in literature, the buffer concentration used for off-line reaction monitoring is at least one order in magnitude lower, which only allows limited comparison of the data.