Shapiro R, Vallee B L
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115.
Biochemistry. 1989 Sep 5;28(18):7401-8. doi: 10.1021/bi00444a038.
The roles of His-13 and His-114 in the ribonucleolytic and angiogenic activities of human angiogenin have been investigated by site-directed mutagenesis. Replacement of either residue by alanine (H13A and H114A) decreases enzymatic activity toward tRNA by at least 10,000-fold and virtually abolishes 10,000-fold and virtually abolishes angiogenic activity in the chick embryo chorioallantoic membrane assay. Both the H13A and H114A mutant proteins compete effectively with angiogenin in the latter assay; only a 5-fold molar excess of H13A over unmodified protein is required for complete inhibition. The His----Ala substitutions, however, do not have any significant effect on the interaction of angiogenin with human placental ribonuclease inhibitor, an extremely potent inhibitor of angiogenin (Ki approximately 7 x 10(-16 M) previously shown to interact with another active-site residue, Lys-40. The effects of more conservative replacements-glutamine at position 13 and asparagine at position 114--were also examined. While the enzymatic activity of the H114N mutant was at least 3300-fold less than for the unmodified protein, the H13Q derivative had only 300-fold reduced activity toward tRNA and cytidylyl(3'----5') adenosine. Both substitutions substantially decreased angiogenic activity. The parallel effects on ribonucleolytic and biological activities observed with all four mutant proteins provide strong evidence that the latter activity of angiogenin is dependent on a functional enzymatic active site. The capacity of the H13A and H114A derivatives to compete with angiogenin in the chorioallantoic membrane assay suggests several additional features of the biological mode of action of this protein.
通过定点诱变研究了组氨酸 -13（His-13）和组氨酸 -114（His-114）在人血管生成素的核糖核酸酶活性和血管生成活性中的作用。用丙氨酸取代这两个残基中的任何一个（H13A和H114A）都会使对tRNA的酶活性降低至少10000倍，并且在鸡胚绒毛尿囊膜试验中几乎完全消除血管生成活性。在后者的试验中，H13A和H114A突变蛋白都能有效地与血管生成素竞争；完全抑制只需H13A相对于未修饰蛋白有5倍的摩尔过量。然而，组氨酸到丙氨酸的取代对血管生成素与人胎盘核糖核酸酶抑制剂的相互作用没有任何显著影响，人胎盘核糖核酸酶抑制剂是血管生成素的一种极其有效的抑制剂（Ki约为7×10⁻¹⁶M），先前已证明它与另一个活性位点残基赖氨酸 -40相互作用。还研究了更保守的取代作用——13位的谷氨酰胺和114位的天冬酰胺。虽然H114N突变体的酶活性比未修饰蛋白至少低3300倍，但H13Q衍生物对tRNA和胞苷酰（3'→5'）腺苷的活性仅降低了300倍。这两种取代都显著降低了血管生成活性。在所有四种突变蛋白中观察到的对核糖核酸酶活性和生物学活性的平行影响提供了有力证据，表明血管生成素的后一种活性依赖于一个功能性的酶活性位点。H13A和H114A衍生物在绒毛尿囊膜试验中与血管生成素竞争的能力表明了这种蛋白质生物学作用模式的几个其他特征。
