TIME-RESOLVED LIGAND-EXCHANGE REACTIONS - KINETIC-MODELS FOR COMPETITIVE INHIBITORS WITH RECOMBINANT HUMAN RENIN

The on and off rate constants (K-on and k(off)) were determined for a series of peptidomimetic, competitive inhibitors of human renin using a novel binding assay. The method entails analyzing a pair of ligand e xchange reactions in which a dansylated inhibitor serves as the fluore scent probe. The first in the pair of reactions involves preincubating renin with the probe and initiating the reaction by addition of a sam ple inhibitor; the second reaction involves preincubating renin with t he sample inhibitor and initiating the reaction by addition of probe. Both reactions yield progress curves which contain complementary infor mation concerning the k(on) and K-off of each ligand. The two curves a re fitted simultaneously using models derived from the differential ra te equations describing the ligand exchange process. The k(on) and k(o ff) rate constants for the probe were 6.85 M(-1) s(-1) and 2.96 x 10(- 4) s(-1), respectively, giving a calculated K-d Of 43.2 pM. The K-d va lues for the inhibitor series varied over 2 orders of magnitude (27-23 20 pM), while the individual k(on) (10(6)-10(7) M(-1) s(-1)) and k(off ) (10(-4)-10(-3) s(-1)) constants varied only over 1 order of magnitud e.