title: Probing intermolecular main chain hydrogen bonding in serine proteinase-protein inhibitor complexes: chemical synthesis of backbone-engineered turkey ovomucoid third domain.
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 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.
authors.affiliation: Scripps Research Institute, La Jolla, California 92037, USA. wuyuan@chemical.ligation.com
authors.forename: W
authors.initials: W
authors.lastname: Lu
authors.forename: M A
authors.initials: MA
authors.lastname: Qasim
authors.forename: M
authors.initials: M
authors.lastname: Laskowski
authors.forename: S B
authors.initials: SB
authors.lastname: Kent
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006860:Hydrogen Bonding; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008958:Models, Molecular; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010059:Ovomucin / Q000138:chemical synthesis* / Q000737:chemistry* / Q000235:genetics; D010084:Oxidation-Reduction; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D012697:Serine Endopeptidases / Q000737:chemistry*; D015842:Serine Proteinase Inhibitors / Q000737:chemistry*; D014422:Turkeys
chemical_list: D046911:Macromolecular Substances; D015842:Serine Proteinase Inhibitors; D010059:Ovomucin; D012697:Serine Endopeptidases