CRYSTAL STRUCTURE OF AN INHIBITOR COMPLEX WITH THE QUINOENZYME AMINE OXIDASE IDENTIFIES THE ACTIVE SITE BASE. Carrie M. Wilmot,Mark R Parsons, Maire A. Convery, Veronica Blakeley, Adam S. Corner, Kapil D.S. Yadav^_, Mike J. McPherson, Peter F. Knowles & Simon E.V. Phillips, Department of Biochemistry and Molecular Biology, The University of Leeds, Leeds, LS2 DJT, United Kingdom_ Present address: Department of Chemistry, University of Gorakhpur, Gorakhpur 273009, India

The crystal structure of the complex between the copper amine oxidase from Escherichia coli (ECAO) and a covalently bound inhibitor, 2-hydrazinopyridine, has been determined to a resolution of 2.5Å. The enzyme contains a cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), which is formed by the post-translational modification of a tyrosine residue. The inhibitor covalently binds at the 5 position of the quinone ring. This complex is analogous to the substrate Schiffs base formed during the reaction. The inhibitor structure has a nitrogen in place of a carbon in the aromatic primary amine substrate, and this prevents the abstraction of a proton by the catalytic base, which would allow the reaction to proceed. The electron density of the Schiffs base moeity in the complex is clear. The inhibitor nitrogen is hydrogen bonded to the sidechain of Asp383, a conserved residue among the known amine oxidase sequences, identifying it as the probable base. The positioning of Asp383 is such that the pro-S proton of a susbtrate would be abstracted. Th TPQ/inhibitor moeity is not directly interacting with the copper. The O4 position on the quinone ring is hydrogen bonded to the axial water ligand of the copper. The O4 position on the quinone ring is involved in a symmetrical hydrogen bond with the hydroxyl of the conserved residue Tyr369. The distance between the oxygens is less thsn 2.5Å, consistent with a shared proton, and suggesting ionisation at the 04 position of the quinone ring. The Tyr369 residue would appear to play an important role in stabilising the position of the quinone/inhibitor complex.