Modulation of the activity of angiogenin by mutagenesis at Asp-116.
Substitution of Asn, Ala or His for Asp-116 in angiogenin increases its ribonucleolytic activity towards tRNA and, at least in the case of His, its ability to induce blood-vessel formation (Harper, J.W. and Vallee, B.L. (1988) Proc. Natl. Acad. Sci. USA 85, 7139-7143). Six additional Asp-116 mutants have been examined to further probe the basis for this phenomenon. Substitution of Val, Lys, Glu, or Ser increases activity towards tRNA 2-, 4-, 9- and 16-fold, respectively, whereas substitution of Trp and Pro leads to 2- and 10-fold decreases, respectively. Similar changes are seen in activity towards rRNA. Studies of base-cleavage specificity towards dinucleotide substrates (NpN') reveal a change in preference for G vs. A at the N' position when Ser replaces Asp-116 and a diminished preference for C vs. U at the N position. The Pro, Lys and Glu mutants have essentially unchanged angiogenic activity. The results demonstrate that the principal effect of replacing Asp-116 in angiogenin is to modulate enzymatic activity, possibly through an effect on His-114, and suggest that Asp-116 plays a role in controlling specificity.
Curran
T P
TP
Center for Biochemical and Biophysical Sciences and Medicine, Boston, MA 02115.
Shapiro
R
R
Riordan
J F
JF
Vallee
B L
BL
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001224:Aspartic Acid; D002642:Chick Embryo; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009389:Neovascularization, Pathologic; D009841:Oligonucleotides; D011506:Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D012260:Ribonucleases / Q000378:metabolism
D000596:Amino Acids; D009841:Oligonucleotides; D011506:Proteins; D001224:Aspartic Acid; D012343:RNA, Transfer; D012260:Ribonucleases; C046366:angiogenin; D012259:Ribonuclease, Pancreatic