Kinetic characterization of two active mutants of placental ribonuclease inhibitor that lack internal repeats.
Human placental ribonuclease inhibitor (PRI), a 50-kDa tight-binding inhibitor of angiogenin and pancreatic ribonuclease, consists predominantly of 7 internal repeats, each 57 residues long. Repeats 3 plus 4 (residues 144-257) or repeat 6 (residues 315-371) can be deleted to give mutant proteins, PRI delta 3-4 and PRI delta 6, respectively, that retain inhibitory activity [Lee, F. S., & Vallee, B. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1879-1883]. We describe here the isolation and characterization of these two active mutant proteins. Both inhibit the enzymatic activities of either angiogenin or bovine pancreatic ribonuclease A (RNase A) with a 1:1 stoichiometry, and the mode of inhibition of RNase A by either is competitive. PRI delta 3-4 binds to angiogenin and RNase A with Ki values of 0.72 and 170 pM, respectively The corresponding values for PRI delta 6 are 22 and 43 pM, respectively. Since recombinant PRI to angiogenin and RNase A with Ki values of 0.29 and 68 fM, respectively, deletion of repeats 3 plus 4 weakens both interactions 2500-fold while deletion of repeat 6 weakens them 76,000- and 630-fold, respectively. Therefore, either the deletion of these repeats has altered the conformation of the angiogenin/RNase binding site in PRI or the deleted repeats contribute directly to the binding site, or both. In addition, the tighter binding to angiogenin versus RNase A seen with native PRI has been preserved in PRI delta 3-4 but has been almost completely abolished in PRI delta 6.(ABSTRACT TRUNCATED AT 250 WORDS)
Lee
F S
FS
Center for Biochemical and Biophysical Sciences, Harvard Medical School, Boston, Massachusetts 02115.
Vallee
B L
BL
D001483:Base Sequence; D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010926:Placental Hormones / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D012259:Ribonuclease, Pancreatic / Q000037:antagonists & inhibitors* / Q000378:metabolism
D010926:Placental Hormones; D011506:Proteins; D011994:Recombinant Proteins; C022052:placental ribonuclease inhibitor; C046366:angiogenin; D012259:Ribonuclease, Pancreatic