A new remote subsite in ribonuclease A

Area of Science:

• Enzymology
• Structural Biology
• Biochemistry

Background:

• Bovine pancreatic ribonuclease A (RNase A) is a key enzyme in RNA degradation.
• Enzyme-substrate interactions involve specific subsites that recognize bases and phosphoryl groups.
• Understanding these interactions is vital for enzyme mechanism studies.

Purpose of the Study:

• To investigate the role of cationic residues in bovine RNase A's interaction with its RNA substrate.
• To identify specific amino acid residues responsible for substrate binding and catalysis.
• To characterize a potential new subsite involved in nucleic acid binding.

Main Methods:

• Site-directed mutagenesis was used to replace four key cationic residues (Lys-37, Arg-39, Arg-85, Lys-104) with alanine.
• Enzymatic assays were performed to measure kinetic parameters (Km, kcat/Km) for poly(cytidylic acid) cleavage.
• Binding affinity (Kd) was determined for enzyme-oligonucleotide complexes at varying salt concentrations.

Main Results:

• Only the replacement of Arg-85 significantly affected both Km and kcat/Km for poly(C) cleavage.
• Arg-85 demonstrated a uniform contribution to both ground and transition state binding, increasing Km by 15-fold and decreasing kcat/Km by 10-fold.
• The salt-dependent binding contribution of Arg-85 indicated a coulombic interaction with a phosphoryl group.

Conclusions:

• Arg-85 plays a critical role in the binding of RNA substrates to bovine RNase A.
• This residue interacts with a specific phosphoryl group, suggesting a distal binding subsite.
• The findings support the proposal of a new P(-1) subsite within RNase A, mediated by Arg-85.