Probing Intermolecular Main Chain Hydrogen Bonding in Serine Proteinase-Protein Inhibitor Complexes: Chemical Synthesis of Backbone-Engineered Turkey Ovomucoid Third Domain. (1996) Lu, W., Qasim, M.A., Laskowski, Jr., and Kent, S.B.H. Biochemistry, 36, No. 4, 673-679.

Abstract

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 CO group of Ser²¹⁴ and Ser¹⁹⁵ O 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.