Identification of functional arginines in human angiogenin by site-directed mutagenesis.
Chemical modifications of human angiogenin had suggested that arginines are essential for its ribonucleolytic activity [Shapiro, R., Weremowicz, S., Riordan, J. F., & Vallee, B. L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8783-8787]. Each of the six arginines within or near angiogenin's catalytic or cell-binding sites--i.e., those at positions 5, 31, 32, 33, 66, and 70--was therefore mutated to alanine. Two of these residues, Arg-5 and Arg-33, indeed play a role, albeit noncrucial, in enzymatic activity, although neither one is implicated in the abolition of activity by arginine reagents. R5A-angiogenin, while nearly fully active toward dinucleotides, is one-fourth as active as angiogenin toward tRNA, suggesting that Arg-5 may participate in the binding of peripheral components of the substrate. In contrast, the activity of R33A-angiogenin toward both polynucleotide and dinucleotide substrates is reduced similarly, reflecting a decrease in kcat. These results, together with its position in the calculated three-dimensional structure of angiogenin, imply an indirect role for Arg-33 in catalysis. Three arginines are important for angiogenesis: mutation of Arg-5, Arg-33, or Arg-66 dramatically reduces the angiogenic potency of angiogenin on the chicken embryo chorioallantoic membrane. Arg-66 lies within a segment previously proposed to be part of a cell-surface receptor binding site. Arg-5 and Arg-33 are outside of this site as defined at present, and the decreased angiogenicity of R5A- and R33A-angiogenin may be a consequence of their reduced ribonucleolytic activities.(ABSTRACT TRUNCATED AT 250 WORDS)
D001120:Arginine / Q000032:analysis* / Q000502:physiology; D001483:Base Sequence; D005798:Genes, Bacterial / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009389:Neovascularization, Pathologic / Q000503:physiopathology*; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000235:genetics; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012259:Ribonuclease, Pancreatic; D012260:Ribonucleases / Q000037:antagonists & inhibitors; D013329:Structure-Activity Relationship
D021382:Protein Sorting Signals; D011506:Proteins; D011993:Recombinant Fusion Proteins; D001120:Arginine; D012260:Ribonucleases; C046366:angiogenin; D012259:Ribonuclease, Pancreatic