The role of non-catalytic 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 main catalytic site, are involved in the formation of the enzyme-substrate complex. This work analyzes the pattern of oligonucleotide formation by ribonuclease A using poly(C) as substrate. The poly(C) cleavage shows that the enzyme does not act in a random fashion but rather prefers the binding and cleavage of the longer substrate molecules and that the phosphodiester bond broken should be 6-7 residues apart from the end of the chain to be preferentially cleaved by ribonuclease A. The results demonstrate the model of the cleavage of an RNA chain based on the cooperative binding between the multisubsite binding structure of ribonuclease A and the phosphates of the polynucleotide (Parés, X., Nogués, M. V., de Llorens, R., and Cuchillo, C. M. (1991) in Essays in Biochemistry (Tipton, K. F., ed) Vol. 26, pp. 89-103, Portland Press Ltd., London). The contribution to the enzymatic process of the non-catalytic phosphate-binding subsite (p2) adjacent to the catalytic center has been analyzed in p2 chemically modified ribonuclease A or by means of site-directed mutagenesis. In both cases deletion of p2 abolishes 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.
Moussaoui
M
M
Departament de Bioquímica i Biologia Molecular, Facultat de Ciències and Institut de Biologia Fonamental V. Villar-Palasí, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain Spain Spain.
Guasch
A
A
Boix
E
E
Cuchillo
C
C
Nogués
M
M
D000495:Allosteric Site; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004722:Endoribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008961:Models, Structural; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000302:isolation & purification; D010179:Pancreas / Q000201:enzymology; D011066:Poly C / Q000378:metabolism; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D013379:Substrate Specificity
D009841:Oligonucleotides; D011066:Poly C; D004722:Endoribonucleases; D012259:Ribonuclease, Pancreatic