Hydrolysis of the cyclic 2',3'-phosphodiester

Figure 3.6: Concentration-rate data for 2',3'-cGMP ester hydrolysis by RNase T1 wild type, obtained using off-line reaction monitoring. From the data acquired, the specificity constant ${}^{k_{cat}}\!\!/\!{}_{K_m}$ could be determined as 0.006  $\left[mMs\right]^{-1}$.
Conditions: 10mM ammonium acetate (pH 6.5), 2',3'-cGMP: 1,900 to 19,000 $\mu$M, RNase T1 890nM, room temperature, n=24

In contrast to the transesterification of the phosphodiester linkage, there is no spectrophotometric assay for the determination of kinetic parameters of the second reaction step of the RNase T1 catalysed cleavage of nucleotides, the hydrolysis of the cyclic phosphodiester. As the second reaction step occurs at a much lower rate than the first one, the second step was monitored separately and the 2',3'-cyclic phosphodiester was used as substrate (substrate concentrations between 6,000 $\mu$M and 19,000 $\mu$M). Due to the low reaction rate, an enzyme concentration of 890 nM was used. Quantification was performed as described in 3.1.3.1, using a calibration curve for the cyclic phosphodiester (see equation 3.4). Due to the low reaction rate, it was impossible to determine MICHAELIS-MENTEN parameters. Instead of this, the specificity parameter ${}^{k_{cat}}\!\!/\!{}_{K_m}$ was acquired by a linear fit of the rate-substrate concentration curve (see figure 3.6). Data for substrate concentrations below 6,000 $\mu$M had to be omitted as the reaction rate could not be determined reliably, even with long-time reaction monitoring for more than twelve hours (data not shown).

The specificity constant ( ${}^{k_{cat}}\!\!/\!{}_{K_m}$) obtained by mass spectrometric off-line reaction monitoring is 0.006  $\left[mMs\right]^{-1}$. Compared with a specificity constant ${}^{k_{cat}}\!\!/\!{}_{K_m}$ of 0.9  $\left[mMs\right]^{-1}$ as described by JAN BACKMANN et al. [6] (see 1.2.1), the value obtained is much lower. However, as mentioned earlier in 3.1.4.1, parameters obtained using different methods and reaction environments are hardly comparable.