Despite Having a Common P₁ Leu, Eglin C Inhibits α-Lytic Proteinase a Million-fold More Strongly than Does Turkey Ovomucoid Third Domain^†

Results of the inhibition of α-lytic proteinase by two standard mechanism serine proteinase inhibitors, turkey ovomucoid third domain (OMTKY3) and eglin C, and many of their variants are presented. Despite similarities, including an identical P₁ residue (Leu) in their primary contact regions, OMTKY3 and eglin C have vastly different association equilibrium constants toward α-lytic proteinase, with K_a values of 1.8 × 10³ and 1.2 × 10⁹ M^-1, respectively. Although 12 of the 13 serine proteinases tested in our laboratory for inhibition by OMTKY3 and eglin C are more strongly inhibited by the latter, the million-fold difference observed here with α-lytic proteinase is the largest we have seen. The million-fold stronger inhibition by eglin C is retained when the K_a values of the P₁ Gly, Ala, Ser, and Ile variants of OMTKY3 and eglin C are compared. Despite the small size of the S₁ pocket in α-lytic proteinase, interscaffolding additivity for OMTKY3 and eglin C holds well for the four P₁ residues tested here. To better understand this difference, we measured K_a values for other OMTKY3 variants, including some that had residues elsewhere in their contact region that corresponded to those of eglin C. Assuming intrascaffolding additivity and using the K_a values obtained for OMTKY3 variants, we designed an OMTKY3-based inhibitor of α-lytic proteinase that was predicted to inhibit 10000-fold more strongly than wild-type OMTKY3. This variant (K¹³A/P¹⁴E/L¹⁸A/R²¹T/N³⁶D OMTKY3) was prepared, and its K_a value was measured against α-lytic proteinase. The measured K_a value was in excellent agreement with the predicted one (1.1 × 10⁷ and 2.0 × 10⁷ M^-1, respectively). Computational protein docking results are consistent with the view that the backbone conformation of eglin C is not significantly altered in the complex with α-lytic proteinase. They also show that the strong binding for eglin C correlates well with more favorable atomic contact energy and desolvation energy contributions as compared to OMTKY3.