Role of glutamine-117 in the ribonucleolytic activity of human angiogenin.
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.
Russo
N
N
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, MA 02115.
Shapiro
R
R
Acharya
K R
KR
Riordan
J F
JF
Vallee
B L
BL
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
D017931:DNA Primers; D011506:Proteins; D005973:Glutamine; D012260:Ribonucleases; C046366:angiogenin; D012259:Ribonuclease, Pancreatic