Active‐Site Covalent Modifications of Quinoprotein Amine Oxidases from Aspergillus niger

Abstract

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