Intermolecular main chain H-bonding networks are frequently encountered at the interface of complexes of protein proteinase inhibitors and their cognate enzymes. Studies of X-ray crystal structures of many protein inhibitors complexed with serine proteinases have revealed that the amide NH group of the P1 residue in the inhibitor donates an H-bond to the carbonyl C = O group of Ser214 and Ser195 Oy in the enzyme (Ser125 and Ser221 in subtilisins, respectively). To probe the energetic contribution of this backbone H-bond in the complexes of OMTKY3 with several serine proteinases, native chemical ligation was used for the total synthesis of a backbone-engineered analog of OMTKY3, in which the amide peptide bond between Thr17 (P2) and Leu18 (P1) was replaced by an ester bond, i.e., -CONH-to-COO-. This chemical "mutation" effectively eliminated the backbone H-bond donated by the NH group of Leu18. By measuring association equilibrium constants for synthetic wild-type OMTKY3 and the backbone-engineered ester analog interacting with a panel of six serine proteinases, we have determined that the P1 NH-->O substitution weakens the binding of OMTKY3 to its cognate enzymes by an average of 15-fold, i.e., 1.5 +/- 0.3 kcal/mol. These results place a quantitative value on the contribution of the intermolecular backbone H-bond in enzyme-inhibitor recognition.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.