Specificity of RNase T1

Table 1.1: Kinetic constants characterising the RNase T1 catalysed cleavage of minimal substrates. a) pH 5.0, 25$^\circ$C, ionic strength: 0.2M [88]; b) pH 7.5, 37$^\circ$C, 50mM Tris-HCl, 1mM EDTA [28]

Substrate	$k_{cat}$	$K_m$	${}^{k_{cat}}\!\!/\!{}_{K_m}$	Reference
	$s^{-1}$	$\mu$M	$M^{-1}s^{-1}$
GpA	84	67	130,000	a
GpC	400	180	220,000	a
GpG	57	37	150,000	a
GpU	33	29	110,000	a
pGpC	1,260	536	240,000	b
ApA			3	a
ApC			5	a
ApG			2	a
ApU			1	a

RNase T1 has a definite preference for a guanosine at its $G_S^B$ site and a cytidine as a leaving group at the $N1_S^B$ position[88] with minimal RNA substrates (e.g. dinucleotide monophosphates). As shown in table 1.1, the catalytic efficiency ( ${}^{k_{cat}}\!\!/\!{}_{K_m}$) is 10$^5$ fold greater for 5',3'-GpN than for the corresponding 5',3'-ApN, and at least 10$^8$ fold greater than corresponding 5',3'-CpN and 5',3'-UpN (data not shown). The higher efficiency and $k_{cat}$ for the pGpC substrate suggests the existence of a $1p$ site. The possibility of an $2p$ site has been supported by binding differences between different dinucleotides[89], but there are no further indications for this. Using site directed mutagenesis to construct certain variants modified in the binding or active site, kinetic studies have been conducted which lead to the ``two-binding-site'' kinetic model (with $G_S^B-p1$ as one site and $N1_S^B$ as the other site)[79]. According to this model, subsite binding energy is converted into chemical activation energy. Structures of different RNaseT1$\cdot$inhibitor complexes, where a putative $N1_S^B$ is occupied, have been reported[51] too. The existence of a $1N_S^B$ has been indicated by binding studies[89,86] and verified and further explored by pH dependent steady-state kinetic studies of RNase T1 [61]. From the results described in the literature so far, an ``optimal'' recognition sequence for RNase T1 can be assumed which is

with intrinsic $-\Delta G$ values for the particular binding sites as shown in table 1.2[87]. Unfortunately, most investigations have been conducted with very short substrates (di- and trinucleotides or analoga), so that there are almost no information considering subsites beyond $2p$ and $N1$. Attempts to use an oligonucleotide with 15 nucleotides have been undertaken[42,43], but the information obtained from these experiments were still insufficient for a further elucidation of subsites and mechanism.

Table 1.2: Free binding energies of the RNase T1 subsites as $-\Delta G$ in ${}^{kJ}\!\!/\!{}_{mol}$ [87]

$G^B$

$\gg$

$G_S$

$\ge$

$2p$

$>$

$1N_S$

$=$

$p1$

$>$

$1N^B$

$>$

$N1$

$-\Delta G$

16.7

4.2

3.9

3.1

3.0

1.8

0