RNase T1 RV

The variant RNase T1 RV was also investigated using CPA. Due to the altered specificity of this enzyme, there are four possible cleavage positions in R/8, which increases the number of possible reaction products. As R/8 was originally designed to investigate an enzyme with guanosine only specificity, not all possible reaction products can be distinguished using mass spectrometry as they have the same molecular weight (e.g. CUAG and GCUA, see figure 3.18 for details).

Figure 3.18: R/8 and its possible RNase T1 RV hydrolysis products with molecular mass (average isotope mass, [M+H]$^+$) and possible intermediates (the first mass represents the molecular weight of the 2',3' cyclic phosphoester, the second one the molecular weight of the final product, the 3' phosphate.)

Table 3.5: Detected cleavage positions of R/8 by RNase T1 RV. The cleavage positions were deduced from the nucleotides detected using MALDI-MS. The bars indicate where hydrolysis by RNase T1 RV took place at the time given. The figures do not distinguish between the cyclic 2',3' phosphodiester and the 3' monophosphate. [E] represents the concentration of RNase T1 RV in nM.
Conditions: 0.91 mM R/8 in water, room temperature; matrix: 3-hydroxypicolinic acid

[E]
10
100

The reaction was conducted with enzyme concentrations between 10 nM and 1,000 nM, the results are shown in table 3.5. The data confirm the presumption, that RNase T1 RV has an altered specificity and does hydrolyse RNA also at the 3' site of an adenosine. However, at a low enzyme concentration, no cleavage products resulting from a hydrolysis of a phosphodiester linkage at the 3' site of adenosine (i.e.cleavage positions $\gamma$ and $\delta$) were detected within the observation time. At an enzyme concentration of 10nM, RNase T1 RV exhibits the same cleavage pattern as RNase T1 wild type, i.e.there is a preference for the hydrolysis at position $\alpha$. At higher enzyme concentrations, a difference appeared between the hydrolysis of the P-O5' phosphoester at the 3' site of adenosine and at the 3' site of guanosine. Whereas the cleavage pattern for the hydrolysis of the P-O5' phosphoester at the 3' site of guanosine is comparable with the cleavage pattern of RNase T1 wild type, the hydrolysis of the P-O5' phosphoester at the 3' site of adenosine occurred mainly at position $\delta$. In all reactions conducted, no products of a hydrolysis at position $\gamma$ could be detected. This supports the presumption, that RNase T1 has a binding site beyond $N1_S^B$, as subtrates with a nucleotide which can bind at $N2_S^B$ are preferred.

For the cleavage at 3' of guanosine, position $\alpha$ with an uracil for position $N2_S^B$ is preferred (see 3.3.2.1) which leads to a higher rate for this reaction. Thus the concentration of nucleotides whith an uncleaved phosphoester linkage at position $\beta$ is higher than for nucleotides with an uncleaved phosphoester linkage at position $\alpha$. The nucleotides resulting from a transesterification at position $\alpha$, i.e.UAG ${}^{c}\!\!/\!{}_{p}$, cannot interact with a possible binding site beyond $N1_S^B$ when adenosine interacts with $G_S^B$. However, this can take place when $G_S^B$ interacts with the adenosine at position $\delta$ and an uncleaved phosphoester linkage at position $\beta$. As the concentration for nucleotides with an uncleaved phosphoester linkage at position $\beta$ is higher than for nucleotides with an uncleaved linkage at position $\alpha$, the $\delta$ cleavage site is preferred by RNase T1 RV in contrast to position $\gamma$, for which less nucleotide with a possible binding site beyond $N1_S^B$ is availabvle.