Binding of placental ribonuclease inhibitor to the active site of angiogenin.
The importance of specific residues in angiogenin for binding to placental ribonuclease inhibitor (PRI) has been assessed by examining the interaction of angiogenin derivatives with PRI. PRI binds native angiogenin with a Ki value of 7.1 X 10(-16) M [Lee, F. S., Shapiro, R., & Vallee, B. L. (1989) Biochemistry 28, 225-230]. Substitution of a Gln for Lys-40 in angiogenin by site-specific mutagenesis decreases the association rate constant 3-fold and increases the dissociation rate constant 440-fold, resulting in a 1300-fold weaker Ki value. The half-life of the mutant.PRI complex is 3.4 h compared to approximately 60 days for the native angiogenin.PRI complex. The magnitude of the change in Ki value suggests that in the complex, Lys-40 forms a salt bridge or hydrogen bond with an anionic moiety in PRI. Carboxymethylation of His-13 or His-114 with bromoacetate increases the Ki value 15-fold, and oxidation of Trp-89 by means of dimethyl sulfoxide and hydrochloric acid increases it 2.4-fold, suggesting that these residues also form part of the contact region with PRI. The changes in Ki value reflect an increase in the dissociation rate constant. On the other hand, dinitrophenylation of either Lys-50 or Lys-60 with 1-fluoro-2,4-dinitrobenzene does not significantly alter the Ki value, suggesting that these residues are not part of the contact region. These results indicate that PRI inhibition minimally involves the three residues critical for the activity of angiogenin--Lys-40, His-13, and His-114--and to a lesser extent its single tryptophan, Trp-89.
Lee
F S
FS
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115.
Vallee
B L
BL
D000596:Amino Acids; D001665:Binding Sites; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009154:Mutation; D010926:Placental Hormones / Q000378:metabolism*; D011247:Pregnancy; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic; D012260:Ribonucleases / Q000037:antagonists & inhibitors*
D000596:Amino Acids; D010926:Placental Hormones; D011506:Proteins; C022052:placental ribonuclease inhibitor; D012260:Ribonucleases; C046366:angiogenin; D012259:Ribonuclease, Pancreatic