title: Direct observation by X-ray analysis of the tetrahedral "intermediate" of aspartic proteinases.
abstract: We report the X-ray analysis at 2.0 A resolution for crystals of the aspartic proteinase endothiapepsin (EC 3.4.23.6) complexed with a potent difluorostatone-containing tripeptide renin inhibitor (CP-81,282). The scissile bond surrogate, an electrophilic ketone, is hydrated in the complex. The pro-(R) (statine-like) hydroxyl of the tetrahedral carbonyl hydrate is hydrogen-bonded to both active-site aspartates 32 and 215 in the position occupied by a water in the native enzyme. The second hydroxyl oxygen of the hydrate is hydrogen-bonded only to the outer oxygen of Asp 32. These experimental data provide a basis for a model of the tetrahedral intermediate in aspartic proteinase-mediated cleavage of the amide bond. This indicates a mechanism in which Asp 32 is the proton donor and Asp 215 carboxylate polarizes a bound water for nucleophilic attack. The mechanism involves a carboxylate (Asp 32) that is stabilized by extensive hydrogen bonding, rather than an oxyanion derivative of the peptide as in serine proteinase catalysis.
authors.affiliation: Department of Crystallography, Birkbeck College, London, UK.
authors.forename: B
authors.initials: B
authors.lastname: Veerapandian
authors.forename: J B
authors.initials: JB
authors.lastname: Cooper
authors.forename: A
authors.initials: A
authors.lastname: Sali
authors.forename: T L
authors.initials: TL
authors.lastname: Blundell
authors.forename: R L
authors.initials: RL
authors.lastname: Rosati
authors.forename: B W
authors.initials: BW
authors.lastname: Dominy
authors.forename: D B
authors.initials: DB
authors.lastname: Damon
authors.forename: D J
authors.initials: DJ
authors.lastname: Hoover
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D006860:Hydrogen Bonding; D008958:Models, Molecular; D009842:Oligopeptides / Q000378:metabolism; D011487:Protein Conformation; D014961:X-Ray Diffraction / Q000379:methods; D000092502:Renin Inhibitors
chemical_list: D001224:Aspartic Acid; D016282:Aspartic Acid Endopeptidases; D009842:Oligopeptides; D000092502:Renin Inhibitors; C074407:CP 81282; C042466:Endothia aspartic proteinase