title: Role of glutamine-117 in the ribonucleolytic activity of human angiogenin.
abstract: The crystal structure of human angiogenin (reported in the preceding paper in this issue) reveals that the site that corresponds to the pyrimidine binding site of RNase A is obstructed by Gln-117. Mutation of this residue to Ala and Gly is here found to increase activity 11- to 18-fold and 21- to 30-fold, respectively, toward dinucleotide, polynucleotide, and cyclic nucleotide substrates, but without changing specificity. The enhanced activity of Q117G toward CpA is due to a 5-fold decrease in Km and a 6-fold increase in kcat. Its Ki value for 2'-CMP is 5-fold lower than that of native angiogenin, whereas its Ki value for 5'-AMP is unchanged. It has been reported previously that mutating Asp-116 to Ala increases activity 15-fold. The double mutant D116A/Q117A is shown to be only slightly more active than each individual mutant. The present results demonstrate that Gln-117 impedes the ribonucleolytic activity of angiogenin, as predicted by x-ray crystallography. Moreover, they suggest that prior to or during catalysis angiogenin must undergo a conformational change to reorient the C-terminal segment that contains this residue, and that a similar reorganization is required for the mutants as well. This view is supported by molecular modeling of an angiogenin-uridine vanadate complex. These in vitro findings have implications for the angiogenic activity of angiogenin in vivo.
authors.affiliation: Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, MA 02115.
authors.forename: N
authors.initials: N
authors.lastname: Russo
authors.forename: R
authors.initials: R
authors.lastname: Shapiro
authors.forename: K R
authors.initials: KR
authors.lastname: Acharya
authors.forename: J F
authors.initials: JF
authors.lastname: Riordan
authors.forename: B L
authors.initials: BL
authors.lastname: Vallee
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000737:chemistry; D005973:Glutamine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic; D012260:Ribonucleases / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D011506:Proteins; D005973:Glutamine; D012260:Ribonucleases; C046366:angiogenin; D012259:Ribonuclease, Pancreatic