Engineered eglin c variants inhibit yeast and human proprotein processing proteases, Kex2 and furin

We engineered eglin c, a potent subtilisin inhibitor, to create inhibitors for enzymes of the Kex2/furin family of proprotein processing proteases. A structural gene was synthesized that encoded "R-1-eglin", having Arg at P-1 in the reactive site loop in place of Leu(45). Ten additional variants were created by cassette mutagenesis of R-1-eglin. These polypeptides were expressed in Escherichia coli, purified to homogeneity, and their interactions with secreted, soluble Kex2 and furin were examined. R-1-eglin itself was a modest inhibitor of Kex2, with a K-a of similar to 10(7) M-1. Substituting Arg (in R4R1-eglin) or Met (in M4R1-eglin) for Pro(42) at P-4 created potent Kex2 inhibitors exhibiting K-a values of similar to 10(9) M-1. R4R1-eglin inhibited furin with a K-a of 4.0 x 10(8) M-1. Introduction of Lys at P-1, in place of Arg in R4R1-eglin reduced affinity only similar to3-fold for Kex2 but 15-fold for furin. The stabilities of enzyme-inhibitor complexes were characterized by association and dissociation rate constants and visualized by polyacrylamide gel electrophoresis. R4R1-eglin formed stable 1:1 complexes with both Kex2 and furin. However, substitution of Lys at P-2 in place of Thr(44) resulted in eglin variants that inhibited both Kex2 and furin but which were eventually cleaved (temporary inhibition). Surprisingly, R6R4R1-eglin, in which Arg was substituted for Gly(40) in R4R1-eglin, exhibited stable, high-affinity complex formation with Kex2 (K-a of 3.5 x 10(9) M-1) but temporary inhibition of furin. This suggests that enzyme-specific interactions can alter the conformation of the reactive site loop, converting a permanent inhibitor into a substrate. Eglin variants offer possible avenues for affinity purification, crystallization, and regulation of proprotein processing proteases.