CATALYTIC MECHANISM OF THE QUINOENZYME AMINE OXIDASE FROM ESCHERICHIA-COLI - EXPLORING THE REDUCTIVE HALF-REACTION

The crystal structure of the complex between the copper amine oxidase from Escherichia coli (ECAO) and a covalently bound inhibitor, 2-hydra zinopyridine, has been determined to a resolution of 2.0 Angstrom. The inhibitor covalently binds at the 5 position of the quinone ring of t he cofactor, 2,4,5-trihydroxyphenylalaninequinone (TPQ). The inhibitor complex is analogous to the substrate Schiff base formed during the r eaction with natural monoamine substrate. A proton is abstracted from a methylene group adjacent to the amine group by a catalytic base duri ng the reaction. The inhibitor, however, has a nitrogen at this positi on, preventing proton abstraction and trapping the enzyme in a covalen t complex. The electron density shows this nitrogen is hydrogen bonded to the side chain of Asp383, a totally conserved residue, identifying it as the probable catalytic base. The positioning of Asp383 is such that the pro-S proton of a substrate would be abstracted, consistent w ith the stereospecificity of the enzyme determined by H-1 NMR spectros copy. Site-directed mutagenesis and in viva suppression have been used to substitute Asp383 for 12 other residues. The resulting proteins ei ther lack or, in the case of glutamic acid, have very low enzyme activ ity consistent with an essential catalytic role for Asp383. The O4 pos ition on the quinone ring is involved in a short hydrogen bond with th e hydroxyl of conserved residue Tyr369. The distance between the oxyge ns is less than 2.5 Angstrom, consistent with a shared proton, and sug gesting ionization at the O4 position of the quinone ring. The Tyr369 residue appears to play an important role in stabilizing the position of the quinone/inhibitor complex. The O2 position on the quinone ring is hydrogen bonded to the apical water Ligand of the copper. The basal water Ligand, which Lies 2.0 Angstrom from the copper in the native s tructure, is at a distance of 3.0 Angstrom in the complex. In the nati ve structure, the active site is completely buried, with no obvious ro ute for entry of substrate. In the complex, the tip of the pyridine ri ng of the bound inhibitor is on the surface of the protein at the edge of the interface between domains 3 and 4, suggesting this as the entr y point for the amine substrate.