Enzymatic hydrolysis of 5',3'-GpC by RNase T1

The enzymatic hydrolysis of 5',3'-GpC by RNase T1 was monitored using the technique described to show its capabilities. To ensure that no autohydrolysis occurs and that the substrate is not contaminated with a ribonuclease, the substrate was monitored without enzyme for at least five minutes. Though no hydrolytic reaction could be detected, small amounts of one or more putative reaction products could be found in almost all substrate solutions which indicates at least a small amount of autohydrolyses during the storage of the substrate or a contamination with the respective products during manufacturing.

Table 3.3: Masses (average isotope mass) of substrates and products of the enzymatic cleavage of the dinucleoside monophosphates 5',3'-GpC and 5',3'-ApC by RNase T1

Compound	Mass	Compound	Mass
5',3'-GpC	588 Da	5',3'-ApC	572 Da
2',3'-cGMP	345 Da	2',3'-cAMP	329 Da
3'-GMP	363 Da	3'-AMP	347 Da
Cytidine	243 Da

The reaction was conducted in 10 mM aqueous ammonium acetate solution (pH 6.5) (to assure a stable spray) inside the spraying capillary. The reaction was started by adding the enzyme to the substrate solution. Immediately thereafter, the enzyme-substrate solution was filled into the spraying capillary using gel loader (microloader) tips, replacing the solution inside the capillary which was used to control autohydrolysis or contamination with ribonucleases.

Figure 3.8: Mass spectrum of the reaction compounds of enzymatic 5',3'-GpC transesterification, recorded using NANOES ten minutes after start of reaction.
Conditions: 1,000 $\mu$M 5',3'-GpC in 10 mM ammonium acetate solution (pH 6.5), room temperature, RNase T1: 2 nM

Figure 3.9: Mass spectrum of a 1 mM cytidine solution. The signals indicated show cytidine and [2$\times$cytidine+H$^+$]$^+$.
Conditions: 10 mM ammonium acetate (pH 6.5), IS 4,200 V; OR 20 V; RNG 295 V; CUR: 8

Figure 3.8 shows a mass spectrum of the reaction compounds ten minutes after the start of the reaction (5',3'-GpC: 1,000 $\mu$M, RNase T1: 2 nM). The spectrum shows the substrate 5',3'-GpC and the products of the first reaction step, cytidine and 2',3'-cGMP. The origin of the ion at 438 m/z could not be resolved. The signal at 487 m/z is the cytidine [2M+H$^+$]$^+$ ion. Further investigations using cytidine showed, that this compound interacts with itself, forming cytinide$\cdot$n complexes, depending on the compound concentration. This could be confirmed using UV spectroscopy. A mass spectrum showing cytidine and cytidine$\cdot$2 is shown in figure 3.9.

Figure 3.10: On-line monitoring of 5',3'-GpC hydrolysis by RNase T1. The figure presents the relative intensities. Data smoothed with a ninth-order Savitzky-Golay filter[72].
Conditions: 2500 $\mu$M 5',3'-GpC in 10 mM ammonium acetate solution (pH 6.5), room temperature, RNase T1 2.7nM

Figure 3.10 shows a time-intensity curve for the enzymatic hydrolysis of 5',3'-GpC by RNase T1 with a substrate concentration of 1,000$\mu$M and an enzyme concentration of 2.7 nM. It can be seen, that within the time monitored, only the first reaction step i.e. the transesterification occurs. This is in accordance with the mechanism proposed by JAN BACKMANN et al. [6], as the second reaction step, the ester hydrolysis, is significantly slower than the transesterification. However, even with monitoring times of 200 minutes under the conditions described, no accumulation of the final reaction product 3'-GMP could be observed. The mass spectrum (see figure 3.11) was extracted at the end of a 200 minute on-line monitoring and showed no 3'-GMP. Even at high enzyme concentrations of 1,000 nM, 3'-GMP is only detectable after a reaction time of 45 minutes at a very low intensity (see spectrum in figure 3.12).

Figure 3.11: Mass spectrum acquired 200 minutes after start of reaction. Despite the long reaction time, no 3'-GMP was detected.
Conditions: 1,000 $\mu$M 5',3'-GpC in 10 mM ammonium acetate solution (pH 6.5), room temperature, RNase T1: 2 nM

Figure 3.12: Mass spectrum acquired 45 minutes after start of reaction with high enzyme concentration. The spectrum shows only a low intensity for 3'-GMP
Conditions: 1,000 $\mu$M 5',3'-GpC in 10mM ammonium acetate solution (pH 6.5), room temperature, RNase T1 1,000nM