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Upstream subsite interactions for oligonucleotide binding with ribonuclease T1
1Department of Chemistry, Kent State University, OH 44242, USA. fwalz@kentvm.kent.edu
Biochimica Et Biophysica Acta
|February 7, 1997
Summary
Ribonuclease T1 binds to RNA substrates through interactions with adenosine and phosphomonoester groups. These binding interactions are pH-dependent and involve specific enzyme subsites for optimal substrate recognition.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Ribonuclease T1 (RNase T1) is known to specifically cleave RNA at guanylyl residues.
- Prior research indicated enzyme-subsite interactions with adenosine residues in specific RNA dinucleotides.
Purpose of the Study:
- To investigate the binding interactions of adenosine-containing dinucleoside monophosphates with RNase T1.
- To elucidate the contribution of the adenosine moiety and its 2'-hydroxyl group to substrate binding affinity.
Main Methods:
- Ultraviolet difference spectroscopy was employed to study the binding of ApG and dApG with RNase T1.
- Binding affinities were analyzed across a pH range of 5-9.
- Thermodynamic parameters (delta G zero, delta delta G) were calculated to quantify binding contributions.
Main Results:
- The binding of adenosine-containing ligands showed pH dependence, influenced by an enzyme/ligand group with a pKa <= 4.8.
- The adenosine moiety contributes approximately -1.17 kcal/mol to the binding free energy of ApG compared to MepG.
- ApG exhibited tighter binding than dApG (delta delta G = -0.73 kcal/mol), indicating the involvement of the adenosine 2'-OH group.
Conclusions:
- RNase T1 possesses specific subsites that accommodate the adenine base and the phosphomonoester group of RNA substrates.
- The adenosine 2'-hydroxyl group plays a significant role in enhancing the binding affinity of RNase T1 for its RNA ligands.
- These findings provide detailed insights into the molecular recognition mechanisms of RNase T1.