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.