THE ROLE OF NONCATALYTIC BINDING SUBSITES IN THE ENDONUCLEASE ACTIVITY OF BOVINE PANCREATIC RIBONUCLEASE-A

Bovine pancreatic ribonuclease A catalyzes the depolymerization of RNA . There is much evidence that several subsites, in addition to the mai n catalytic site, are involved in the formation of the enzyme-substrat e complex. This work analyzes the pattern of oligonucleotide formation by ribonuclease A using poly(C) as substrate. The poly(C) cleavage sh ows that the enzyme does not act in a random fashion but rather prefer s the binding and cleavage of the longer substrate molecules and that the phosphodiester bond broken should be 6-7 residues apart from the e nd of the chain to be preferentially cleaved by ribonuclease A. The re sults demonstrate the model of the cleavage of an RNA chain based on t he cooperative binding between the multisubsite binding structure of r ibonuclease A and the phosphates of the polynucleotide (Pares, X., Nog ues, M. V., de Llorens, R., and Cuchillo, C. M. (1991) in Essays in Bi ochemistry (Tipton, R. F., ed) Vol. 26, pp. 89-103, Portland Press Ltd ., London). The contribution to the enzymatic process of the non-catal ytic phosphate-binding subsite (p(2)) adjacent to the catalytic center has been analyzed in p(2) chemically modified ribonuclease A or by me ans of site-directed mutagenesis. In both cases deletion of p(2) aboli shes the endonuclease activity of ribonuclease A, which is substituted by an exonuclease activity. All these results support the role of the multisubsite structure of the enzyme in the endonuclease activity and in the catalytic mechanism.