Despite Having a Common P1 Leu, Eglin C Inhibits α-Lytic Proteinase a Million-fold More Strongly than Does Turkey Ovomucoid Third Domain†
datasetposted on 2006-09-26, 00:00 authored by M. A. Qasim, Robert L. Van Etten, Tina Yeh, C. Saunders, P. J. Ganz, S. Qasim, L. Wang, M. Laskowski
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 Ka 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 Ka 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 Ka 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 Ka 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 Ka value was measured against α-lytic proteinase. The measured Ka 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.