title: Contribution of Tyr712 and Phe716 to the activity of human RNase L.
abstract: Ribonuclease L (RNase L) is a key enzyme in the 2-5A host defense system, and its activity is strictly regulated by an unusual 2',5'-linked oligoadenylate (2-5A). A bipartite model, in which the N-terminal half of RNase L is responsible for the 2-5A binding and the C-terminal half alone is able to hydrolyse the substrate RNA, has been proposed on the basis of the results of deletion mutant analyses [Dong, B. & Silverman, R.H. (1997) J. Biol. Chem.272, 22236-22242]. Above all, the region between Glu711 and His720 was revealed to be essential for RNA binding and/or hydrolysis. To dissect the function of the region, we performed scanning mutagenesis over the 10 residues of glutathione S-transferase (GST)-fusion RNase L. Among the single amino acid mutants examined, Y712A and F716A resulted in a significant decrease of RNase activity with a reduced RNA binding acitivity. The losses of the RNase activity were not restored by its conservative mutation, whereas the RNA binding activity was enhanced in the case of Y712F. These results indicate that both Tyr712 and Phe716 provide the enzyme with a RNA binding activity and catalytic environment.
authors.affiliation: Laboratory of Molecular Biochemistry, Department of Biomolecular Science, Faculty of Engineering, Gifu University, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Nakanishi
authors.forename: Akihiro
authors.initials: A
authors.lastname: Yoshimura
authors.forename: Norihisa
authors.initials: N
authors.lastname: Ishida
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Ueno
authors.forename: Yukio
authors.initials: Y
authors.lastname: Kitade
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D004722:Endoribonucleases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000378:metabolism*; D010957:Plasmids; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D014443:Tyrosine; D010649:Phenylalanine; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease