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 E-amino group of the Lys356 residue in the sequence Lys-Met-Pro-Asn-Ala of Aspergillus niger amine oxidase AO-I.