title: 'pH-jump' crystallographic analyses of gamma-lactam-porcine pancreatic elastase complexes.
abstract: beta-Lactams inhibit a range of enzymes via acylation of nucleophilic serine residues. Certain gamma-lactam analogues of monocyclic beta-lactams have also been shown to be reversible inhibitors of porcine pancreatic elastase (PPE), forming acyl-enzyme complexes that are stable with respect to hydrolysis. Crystallographic analysis at pH 5 of an acyl-enzyme complex formed with PPE and one of these inhibitors revealed the ester carbonyl located in the oxyanion hole in a similar conformation to that observed in the structure of a complex formed between a heptapeptide (beta-casomorphin-7) and PPE. Only weak electron density was observed for the His-57 side chain in its 'native' conformation. Instead, the His-57 side chain predominantly adopted a conformation rotated approx. 90 degrees from its normal position. PPE-gamma-lactam crystals were subjected to 'pH-jumps' by placing the crystals in a buffer of increased pH prior to freezing for data collection. The results indicate that the conformation of the gamma-lactam-derived acyl-enzyme species in the PPE active site is dependent on pH, a result having implications for the analysis of other serine protease-inhibitor structures at non-catalytic pH values. The results help to define the stereoelectronic relationship between the ester of the acyl-enzyme complex, the side chain of His-57 and the incoming nucleophile during the reversible (de)acylation steps, implying it is closely analogous to the hydrolytic deacylation step during catalytic peptide hydrolysis.
authors.affiliation: The Oxford Centre for Molecular Sciences and The Dyson Perrins Laboratory, South Parks Road, Oxford OX1 3QY, UK.
authors.forename: P A
authors.initials: PA
authors.lastname: Wright
authors.forename: R C
authors.initials: RC
authors.lastname: Wilmouth
authors.forename: I J
authors.initials: IJ
authors.lastname: Clifton
authors.forename: C J
authors.initials: CJ
authors.lastname: Schofield
mesh_terms: D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D004583:Electrons; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D007769:Lactams / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D010196:Pancreatic Elastase / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000378:metabolism; D013552:Swine; D013997:Time Factors
chemical_list: D007769:Lactams; D006639:Histidine; D012697:Serine Endopeptidases; D010196:Pancreatic Elastase