Reaction mechanism

Figure 1.2: Reaction mechanism of RNase T1 according to JAN BACKMANN et al. [6]. Top row: hydrolysis of the 5',3'-phosphodiester linkage; bottom row: protonation state of Glu58 and His92

Though the internucleotide phosphodiester linkage of RNA is stable, it is vulnerable to acid- or alkali-promoted hydrolysis. The proximity of the adjacent 2'-hydroxyl group to the phosphorous centre of the internucleotide linkage permits a facile transesterification, in particular under strong acidic or strong basic conditions. Both, acid- and alkali-promoted mechanisms proceed via a type two nucleophilic substitution ($S_N2$) and lead to a 2',3'-cyclic phosphodiester and a 5'-hydroxyl terminus[52]. This cyclising mechanism for RNA cleavage, which was first proposed and subsequently established during the 1950s[9], is the primary pathway for the uncatalysed degradation of RNA polymers under cellular conditions. The resulting intermediate, the 2',3'-cyclic monophosphate, is hydrolysed in absence of an enzyme to form a mixture of nucleoside 2'- and 3'-phosphates. The product distribution is 1:2 in favour of the 3' products under acidic and neutral conditions, whereas it is more even in alkaline solutions, though the 3'-phosphate still prevails. Under the conditions described, the rates for cyclophosphodiester cleavage are at least one order of magnitude higher than for the cleavage of the internucleotide phosphodiester linkage. Hence, the observation of the cyclic phosphodiester is difficult in acid- or alkali-promoted RNA hydrolysis[59].

In contrast to the catalysis via BRØNSTED acids and bases, the intermediate 2',3'-cyclic phosphodiester accumulates in the reaction catalysed by RNase T1, and is cleaved subsequently at a rate much lower than the transesterification ( ${}^{k_{cat}}\!\!/\!{}_{K_m}$ ( $\left[mMs\right]^{-1}$): 1,930 vs. 0.9[6]) to only form the 3'-monophosphate. The transesterification consists of a nucleophilic substitution at the phosphorus atom of the 5'-O leaving group by the incoming 2'-oxygen atom.

Despite the fact that RNase T1 is a well characterised enzyme, the reaction mechanism is still not resolved en detail[4]. Nevertheless it is known, that Glu58 acts as a general base and the $\beta$-imidazolium group of His92 acts as a general acid in a concerted action for enzyme catalysed transesterification[6], whereas the side chains of Tyr38, Arg77 and Phe100 are mainly involved in the electrostatic stabilisation of the transition state, the trigonal bipyramidal phosphorane (TBP)[78]. After the first reaction step (the transesterification), the proton donor His92 loses its proton, whereas the proton acceptor Glu58 becomes protonated. In this state, the enzyme is incapable of performing the transesterification of another 5',3'-phosphodiester linkage. In the second reaction step (the ester hydrolysis), the enzyme becomes transformed back to the starting point. In this reaction step, the cyclic 2',3'-phosphodiester is hydrolysed by a substitution at the 2'-oxygen by OH. In this reaction, Glu58 acts as proton donor and His92 as acceptor[31]. This mechanism was confirmed e.g. by time resolved crystallography with a slow substrate[97]. According to this mechanism, it could be assumed that both steps have similar rates. However, JAN BACKMANN et al. found by extended kinetic analysis of RNase T1 (wild type and selected variants) a ``short cut'' for the second reaction step which uncouples both steps[6]. In this ``short cut'', water acts as ``substrate'' and performs the hydrogen transfer between the active residues. This ``short cut'' is preferred compared with the ester hydrolysis, which results in a higher rate for the first reaction step. It is assumed, that this is due to the inability of the cyclic 2',3'-guanosine monophosphate for subsite interactions. Furthermore, the equilibrium of the water mediated proton exchange between Glu58 and His92 is shifted far to the left[6].

Figure 1.3: Binding of the guanosine base moiety in the binding site of RNase T1. Structure data from 1RNT [3], generated with the WebLabViewer software package (WebLabViewer Lite, Molecular Simulat. Inc, USA) and VMD [36]. Only hydrogens necessary for binding are shown. Side chains are omitted unless interacting with the substrate, except for the two stacking amino acids (Tyr42 and Tyr45), which have been omitted too.

The substrate recognition is performed via a loop of the residues Tyr42 to Glu46 and Asn98, as shown in figure 1.3. Guanosine specificity originates from hydrogen bonds between the guanine base moiety, the peptide backbone (Asn43, Asn44, Tyr45 and Asn98) and the $\delta$-carboxy group of Glu46. Furthermore, the sandwich-like parallel stacking of the guanosine base between the phenolic Tyr42 and Tyr45 [56,64] fixes the substrate. Kinetic investigations[60,88] showed that the leaving group (i.e.N in 5',3'-GpN) has also an influence on the enzymatical catalysis, moreover, the rate limiting step of the transesterification depends on the nature of N[80].

To describe the enzyme subsites which interact with the RNA moieties, the following nomenclature, which was proposed by FREDERIC WALZ[89,87] was used in this work:

In this nomenclature, a capital $N$ represents a nucleoside and a $p$ a poshodiester linkage. The superscript $^B$ represents thenucleobase part of the nucleotide, the subscript $_S$ the ribose (sugar) part. The guanosine binding site is labelled with $G$. Thus the primary recognition site of RNase T1 which binds guanosine and its phosphate group is denoted as $G_S^B-p1$, whereas the leaving group of a dinucleoside monophosphate is denoted as $N1_S^B$. The remaining $p$ and $N_S^B$ moities designate other possible binding sites.