RNase T1 wild type

The cleavage pattern of R/8 by RNase T1 wild type was investigated. The enzymatic reaction was conducted in aqueous solution to reduce the amount of salt ions interferring with the MALDI. An enzyme concentration as low as possible for the reaction to take place in a suitable time was used to ensure that all intermediates of the hydrolysis could be detected. Before starting the reaction, a sample was taken from each reaction vessel as a control; a spectrum of R/8 is shown in 3.17. The reaction was started by adding 1 $\mu$l of enzyme solution to 10 $\mu$l substrate solution (0.1 mM). Immediately after the start of the reaction, samples of 0.5 $\mu$l were taken from the reaction vessel, mixed with the same amount of matrix solution (see 2.2.2.3) on the sample plate and dried at room temperature. The samples were investigated using MALDI-TOF-MS with the method described in 2.2.2.3. To enhance sensitifity, the low mass gate was set to 500 Da. Nevertheless, all products except for cytidine could be detected. The peaks were assigned to the particular cleavage products according to figure 3.16.

Table 3.4: Detectable cleavage products of the enzymatic hydrolysis of R/8 by RNase T1 wild type. The first column represents the concentration of RNase T1 wt in nM. The coloured bars indicate the nucleotides which have been be detected by MALDI-MS after the reaction time given. The coloured bars represent the following nucleotides (sequences from 5' to 3', the figures do not distinguish between the 2',3' cyclic phosphate and the 3' monophosphate): the red bar R/8, the cyan bar UAG ${}^{c}\!\!/\!{}_{p}$, the green bar CUAGC, the purple bar UAGCUAG ${}^{c}\!\!/\!{}_{p}$.
Conditions: 0.91 mM R/8 in water, room temperature; matrix: 3-hydroxypicolinic acid

0.1
1.0
100.0

The reaction was conducted with RNase T1 wild type concentrations between 0.1nM and 1,000nM. The results of these investigations are shown in table 3.4. There are several inconsistencies regarding the detected products, e.g. the solitary occurrence of CUAGC (hydrolysis at $\alpha$), which should be normaly accompanied by UAG ${}^{c}\!\!/\!{}_{p}$ ( ${}^{c}\!\!/\!{}_{p}$ represents either the 2',3' cyclic phosphodiester (p) or the 3' monophosphate). However, this may be due to the different properties of the substrate regarding ionisation efficiency. The data show a preference of RNase T1 wild type regarding the position of the cleavage sequence. Cleavage products, which result in a hydrolysis at position $\alpha$ occur much earlier than the cleavage products of position $\beta$. There are two possible explanations for this; first, RNase T1 may prefer cleavage positions which are embedded into a longer nucleotide sequence in both, 5' and 3' direction. The second possibility is that RNase T1 has a post $N1$ recognition site which has an influence on the reaction rate.

Furthermore, the data obtained support the mechanism proposed by JAN BACKMANN et al. [6], as the 3' phosphates were detectable only at high enzyme concentrations and long reaction times. UAGp, the 3' monophosphate was only detectable at enzyme concentrations larger than 10 nM and a reaction time of more than nine minutes. Similar data were found for the hydrolysis of the cyclic 2',3' phosphodiester at position $\beta$. These findings indicate, that substrates longer than dinucleotides also dissociate from the enzyme after the first reaction step, as otherwise no intermediate would have been detectable. Thus there seems to be only a weak interaction between RNase T1 wild type and a small oligonucleotide with the sequence of R/8.