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Probing Intermolecular Main Chain Hydrogen Bonding in Serine Proteinase−Protein Inhibitor Complexes: Chemical Synthesis of Backbone-Engineered Turkey Ovomucoid Third Domain

1997; American Chemical Society; Volume: 36; Issue: 4 Linguagem: Inglês

10.1021/bi9625612

1943-295X

Wuyuan Lu, Mohammad Qasim, Michael Laskowski, Stephen B. H. Kent,

Enzyme Production and Characterization

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 Oγ 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.