Two engineered eglin c mutants potently and selectively inhibiting kexin or furin

Abstract

Eglin c with mutants L45R and D42R at the P₁ and P₄ positions has been reported to become a stable inhibitor toward the proprotein convertases (PC), furin and kexin, with a K _i of 2.3×10⁻⁸ and 1.3×10⁻¹⁰ M, respectively. The mutant was further engineered at the P₂′–P₄′ positions to create a more potent and selective inhibitor for each enzyme. The residue Asp at P₁′ which is crucial for stabilizing the conformation of eglin c remained unchanged. The eglin c mutants cloned into the vector pGEX‐2T and expressed in Escherichia coli (DH5α) were purified to homogeneity, and their inhibitory activities toward the purified recombinant furin and kexin were examined. The results showed that (1) Leu47 at P₂′ replaced with either a positively or negatively charged residue resulted in a decrease in inhibitory activities to both enzymes; (2) the replacement of Arg with Asp at P₃′ was favorable for inhibiting furin with a K _i of 7.8×10⁻⁹ M, but not for inhibiting kexin; (3) the replacement of Tyr with Glu at P₄′ increased the inhibitory activity to kexin with a K _i of 3×10⁻¹¹ M, but was almost without any influence on furin inhibition. It was indicated that the inhibitory specificity of eglin c could be changed from inhibiting elastase to inhibiting PCs by site‐directed mutation at the P positions, while the inhibitory selectivity to furin or kexin could be optimized by mutation at the P′ positions.