Kinetic characterization of two active mutants of placental ribonuclease inhibitor that lack internal repeats

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Abstract
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., .ALPHA.MP 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 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 delection of repeat 6 weakens them 76000-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. 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 .cntdot. native PRI complex is also present in both mutant complexes: Like native PRI, PRI.DELTA.3-4 and PRI.DELTA.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.