Structural insights into the substrate specificity of bacterial copper amine oxidase obtained by using irreversible inhibitors

• Published in: Journal of biochemistry (Tokyo) Vol. 151; no. 2; pp. 167 - 178
• Main Authors: Murakawa, Takeshi, Hayashi, Hideyuki, Taki, Masayasu, Yamamoto, Yukio, Kawano, Yoshiaki, Tanizawa, Katsuyuki, Okajima, Toshihide
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01-02-2012
• Subjects: Amine Oxidase (Copper-Containing) - antagonists & inhibitors; Amine Oxidase (Copper-Containing) - chemistry; Amine Oxidase (Copper-Containing) - metabolism; Arthrobacter - enzymology; Arthrobacter - metabolism; Arthrobacter globiformis; Bacteria; Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Binding Sites; Catalysis; Dihydroxyphenylalanine - analogs & derivatives; Dihydroxyphenylalanine - chemistry; Dihydroxyphenylalanine - metabolism; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - metabolism; Hydrazines - chemistry; Hydrazines - metabolism; Hydrolysis; Kinetics; Oxidation-Reduction; Schiff Bases; Substrate Specificity; topa quinone; irreversible inhibitor; X-ray crystal structure; copper amine oxidase; substrate specificity
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/jb/mvr125

Copper amine oxidases (CAOs) catalyse the oxidation of various aliphatic amines to the corresponding aldehydes, ammonia and hydrogen peroxide. Although CAOs from various organisms share a highly conserved active-site structure including a protein-derived cofactor, topa quinone (TPQ), their substrate specificities differ considerably. To obtain structural insights into the substrate specificity of a CAO from Arthrobacter globiformis (AGAO), we have determined the X-ray crystal structures of AGAO complexed with irreversible inhibitors that form covalent adducts with TPQ. Three hydrazine derivatives, benzylhydrazine (BHZ), 4-hydroxybenzylhydrazine (4-OH-BHZ) and phenylhydrazine (PHZ) formed predominantly a hydrazone adduct, which is structurally analogous to the substrate Schiff base of TPQ formed during the catalytic reaction. With BHZ and 4-OH-BHZ, but not with PHZ, the inhibitor aromatic ring is bound to a hydrophobic cavity near the active site in a well-defined conformation. Furthermore, the hydrogen atom on the hydrazone nitrogen is located closer to the catalytic base in the BHZ and 4-OH-BHZ adducts than in the PHZ adduct. These results correlate well with the reactivity of 2-phenylethylamine and tyramine as preferred substrates for AGAO and also explain why benzylamine is a poor substrate with markedly decreased rate constants for the steps of proton abstraction and the following hydrolysis.