KINETIC CHARACTERIZATION OF 2 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∆3-4 and PRI∆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. PR1∆3-4 binds to angiogenin and RNase A with Ki values of 0.72 and 170 pM, respectively. The cor responding values for PRI∆6 are 22 and 43 pM, respectively. Since recombinant PRI binds 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∆3-4 but has been almost completely abolished in PRI∆6. Despite the substantial weakening of affinity observed for these mutant proteins, a critical contact with the active-site Lys-40 of angiogenin that is present in the angiogenin-native PRI complex is also present in both mutant complexes: like native PRI, PRI∆3-4 and PRI∆6 bind substantially more weakly, 9200- and 6800-fold, respectively, to an angiogenin mutant in which Lys-40 has been changed to Gln than to native angiogenin. © 1990, American Chemical Society. All rights reserved.