THE CONTRIBUTION OF NONCATALYTIC PHOSPHATE-BINDING SUBSITES TO THE MECHANISM OF BOVINE PANCREATIC RIBONUCLEASE-A

The enzymatic catalysis of polymeric substrates such as proteins, poly saccharides or nucleic acids requires precise alignment between the en zyme and the substrate regis flanking the region occupying the active site. In the case of ribonucleases, enzyme-substrate binding may be di rected by electrostatic interactions between the phosphate groups of t he RNA molecule and basic amino acid residues on the enzyme. Specific interactions between the nitrogenated bases and particular amino acids in the active site or adjacent positions may also take place. The sub strate-binding subsites of ribonuclease A have been characterized by s tructural and kinetic studies. In addition to the active site (p(1)), the role of other noncatalytic phosphate-binding subsites in the corre ct alignment of the polymeric substrate has been proposed, p(2) and p( 0) have been described as phosphate-binding subsites that bind the pho sphate group adjacent to the 3' side and 5' side, respectively, of the phosphate in the active site. In both cases, basic amino acids (Lys-7 and Arg-10 in p(2), and Lys-66 in p(0)) are involved in binding. Howe ver, these binding sites play different roles in the catalytic process of ribonuclease A. The electrostatic interactions in p(2) are importa nt both in catalysis and in the endonuclease activity of the enzyme, w hilst the p(0) electrostatic interaction contributes only to binding o f the RNA.