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 Aspergil lus niger, AO-I and AO-II, with active-site covalent modifiers have be en investigated. Both enzymes are inhibited similarly by phenylhydrazi ne or p-nitrophenylhydrazine, forming an orange Schiff base with a car bonyl group of topaquinone cofactor. Modification of histidyl and tyro syl residues by diethylpyrocarbonate and sulfhydryl groups by 5,5'-dit hio-bis-(2-nitrobenzoic acid) and 4-chloro-7-nitrobenzo-2-oxa-1,3-diaz ole have been described. A substrate analog, 1,4-diamino-2-butyne was found to function as a mechanism-based inhibitor. It shows both substr ate saturation kinetics and time-dependent irreversible inhibition cau sed by formation of pyrrole bound to the active site. The pyrrole form ation was confirmed spectrophotometrically by reaction with Ehrlich's reagent at 525 nm. Inhibition by 1,4-diamino-2-butyne produces a new m aximum in the absorption spectra of AO-I and AO-II at 310 nm and 306 n m, respectively. Inactivated AO-I was digested by proteases; labeled p eptides were purified by C18 HPLC and sequenced by Edman degradation. Data reveal the evidence that 1,4-diamino-2-butyne reacts with the E-a mino group of the Lys356 residue in the sequence Lys-Met-Pro-Asn-Ala o f Aspergillus niger amine oxidase AO-I.