Active-site covalent modifications of quinoprotein amine oxidases from Aspergillus niger. Evidence for binding of the mechanism-based inhibitor, 1,4-diamino-2-butyne, to residue Lys356 involved in the catalytic cycle.
Interactions of two distinct quinoprotein amine oxidases from Aspergillus niger, AO-I and AO-II, with active-site covalent modifiers have been investigated. Both enzymes are inhibited similarly by phenylhydrazine or p-nitrophenylhydrazine, forming an orange Schiff base with a carbonyl group of topaquinone cofactor. Modification of histidyl and tyrosyl residues by diethylpyrocarbonate and sulfhydryl groups by 5,5'-dithio-bis-(2-nitrobenzoic acid) and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole have been described. A substrate analog, 1,4-diamino-2-butyne, was found to function as a mechanism-based inhibitor. It shows both substrate saturation kinetics and time-dependent irreversible inhibition caused by formation of pyrrole bound to the active site. The pyrrole formation was confirmed spectrophotometrically by reaction with Ehrlich's reagent at 525 nm. Inhibition by 1,4-diamino-2-butyne produces a new maximum in the absorption spectra of AO-I and AO-II at 310 nm and 306 nm, respectively. Inactivated AO-I was digested by proteases; labeled peptides were purified by C18 HPLC and sequenced by Edman degradation. Data reveal the evidence that 1,4-diamino-2-butyne reacts with the epsilon-amino group of the Lys356 residue in the sequence Lys-Met-Pro-Asn-Ala of Aspergillus niger amine oxidase AO-I.
Frébort
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I
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan.
Pec
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P
Luhová
L
L
Matsushita
K
K
Toyama
H
H
Adachi
O
O
D006631:Amine Oxidase (Copper-Containing) / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D003959:Diamines / Q000378:metabolism / Q000494:pharmacology; D004047:Diethyl Pyrocarbonate / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010659:Phenylhydrazines / Q000378:metabolism; D012545:Schiff Bases / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013379:Substrate Specificity
D003959:Diamines; D010659:Phenylhydrazines; D012545:Schiff Bases; C030299:phenylhydrazine; C005307:4-nitrophenylhydrazine; C063335:1,4-diamino-2-butyne; D006631:Amine Oxidase (Copper-Containing); D008239:Lysine; D004047:Diethyl Pyrocarbonate