title: Upstream subsite interactions for oligonucleotide binding with ribonuclease T1.
abstract: Ribonuclease T1 (RNase T1) 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 T1 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 pKa value of < or = 4.8. delta G zero for ApG binding minus that for MepG at pH > 6 yielded a delta delta G 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 delta delta G 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 delta delta G for maximal binding the adenine base per se was -0.44 kcal/mol. delta delta G for binding pdApdG minus that for dApdG (-0.94 kcal/mol) suggested an enzyme subsite for the phosphomonoester group of former ligand.
authors.affiliation: Department of Chemistry, Kent State University, OH 44242, USA. fwalz@kentvm.kent.edu
authors.forename: F G
authors.initials: FG
authors.lastname: Walz
mesh_terms: D000241:Adenosine / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D006163:Ribonuclease T1 / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
chemical_list: D008024:Ligands; D009841:Oligonucleotides; D006163:Ribonuclease T1; D000241:Adenosine