Catalytic Mechanism of the Quinoenzyme Amine Oxidase from Escherichia coli: Exploring the Reductive Half-Reaction
详细信息 查看全文
- 作者：Carrie M. Wilmot ; Jeremy M. Murray ; Gordon Alton ; Mark R. Parsons ; Maire A. Convery ; Veronica Blakeley ; Adam S. Corner ; Monica M. Palcic ; Peter F. Knowles ; Michael J. McPherson ; and Simon E. V. Philli
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：February 18, 1997
- 年：1997
- 卷：36
- 期：7
- 页码：1608 - 1620
- 全文大小：314K
- 年卷期：v.36,no.7(February 18, 1997)
- ISSN：1520-4995
文摘
The crystal structure of the complex between the copper amineoxidase from Escherichia coli(ECAO) and a covalently bound inhibitor, 2-hydrazinopyridine, has beendetermined to a resolution of2.0 Å. The inhibitor covalently binds at the 5 position of thequinone ring of the cofactor,2,4,5-trihydroxyphenylalaninequinone (TPQ). The inhibitor complexis analogous to the substrate Schiffbase formed during the reaction with natural monoamine substrate.A proton is abstracted from a methylenegroup adjacent to the amine group by a catalytic base during thereaction. The inhibitor, however, has anitrogen at this position, preventing proton abstraction and trappingthe enzyme in a covalent complex.The electron density shows this nitrogen is hydrogen bonded to theside chain of Asp383, a totally conservedresidue, identifying it as the probable catalytic base. Thepositioning of Asp383 is such that the pro-Sproton of a substrate would be abstracted, consistent with thestereospecificity of the enzyme determinedby 1H NMR spectroscopy. Site-directed mutagenesis andin vivo suppression have been used tosubstituteAsp383 for 12 other residues. The resulting proteins either lackor, in the case of glutamic acid, havevery low enzyme activity consistent with an essential catalytic rolefor Asp383. The O4 position on thequinone ring is involved in a short hydrogen bond with the hydroxyl ofconserved residue Tyr369. Thedistance between the oxygens is less than 2.5 Å, consistent with ashared proton, and suggesting ionizationat the O4 position of the quinone ring. The Tyr369 residue appearsto play an important role in stabilizingthe position of the quinone/inhibitor complex. The O2 position onthe quinone ring is hydrogen bondedto the apical water ligand of the copper. The basal water ligand,which lies 2.0 Å from the copper in thenative structure, is at a distance of 3.0 Å in the complex. Inthe native structure, the active site is completelyburied, with no obvious route for entry of substrate. In thecomplex, the tip of the pyridine ring of thebound inhibitor is on the surface of the protein at the edge of theinterface between domains 3 and 4,suggesting this as the entry point for the aminesubstrate.