INTERSCAFFOLDING ADDITIVITY - ASSOCIATION OF P-1 VARIANTS OF EGLIN-C AND OF TURKEY OVOMUCOID 3RD DOMAIN WITH SERINE PROTEINASES

Standard mechanism protein inhibitors of serine proteinases share a co mmon mechanism of interaction with their cognate enzymes. The P-1 resi due of the inhibitor interacts with the enzyme in a substrate-like man ner. Its side chain becomes imbedded in the S-1 cavity of the enzyme. The nature of P-1, the primary specificity residue, greatly affects th e strength and specificity of the enzyme inhibitor association. In can onical inhibitors, residues P-4-P-2'(P-3'), where P-1-P-1' is the reac tive site, share a common main chain conformation that does not change on complex formation. The remainder of the inhibitor's structure, the scaffolding, is not always common. Instead, there are at least 20 inh ibitor families, each with a different scaffolding. In this paper, we ask whether the differences in standard free energy of association of enzyme-inhibitor complexes upon P-1 mutations are independent of the n ature of the scaffolding. We have already reported on 25 P-1 variants of turkey ovomucoid third domain, a member of the Kazal inhibitor fami ly, interacting with six different serine proteinases. Here, we report on seven different P-1 variants of eglin c, a potato I family member, interacting with the same six serine proteinases under the same condi tions. The differences in standard free energy on P-1 mutations in the eglin c system agree very well, when P-1 Pro is omitted. Complete agr eement indicates that these P-1 residues are interscaffolding additive . This is consistent with the superimposition of the high-resolution s tructures of eglin c and of turkey ovomucoid third domain with chymotr ypsin. In both cases, the P-1 Leu side chain is similarly oriented in almost indistinguishable specificity pockets of the enzyme.