Upstream subsite interactions for oligonucleotide binding with ribonuclease T 1

Abstract

Ribonuclease T 1 (RNase T 1) specifically cleaves RNA at guanylyl residues. Previous studies revealed the presence of an enzyme-subsite interaction for adenosine residues of ApGpC and ApGpU substrates (Osterman and Walz (1979) Biochemistry 10, 1984–1988). The binding of ApG and 2′-deoxyadenylyl-(3′,5′)-guanosine (dApG) with RNase T 1 was studied in the pH range 5–9 using ultraviolet difference spectroscopy. The association constants for these dinucleoside monophosphates showed the same pH dependence both of which differed at low pH values with that for the methyl phosphoester of 5′-GMP (MepG). This difference suggested that binding of the adenosine group is strongly dependent on the deprotonation of an enzyme/ligand group with a p K a value of ≤4.8. ΔG° for ApG binding minus that for MepG at pH>6 yielded a Δ ΔG of −1.17±0.10 kcal/mol which is a measure of the contribution of the adenosine moiety to binding. ApG bound more tightly than dApG with a mean Δ ΔG value of −0.73±0.10 kcal/mol which demonstrated the involvement of the adenosine 2′-OH group in binding. These and other comparisons indicated that Δ ΔG for maximal binding the adenine base per se was −0.44 kcal/mol. Δ ΔG for binding pdApdG minus that for dApdG (−0.94 kcal/mol) suggested an enzyme subsite for the phosphomonoester group of former ligand.