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-11, 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-l,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-I1 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 1P-diamino-2-butyne reacts with the &-amino group of the Lys356 residue in the sequence Lys-Met-Pro-AsnAla of Aspergillus niger amine oxidase AO-I.Amine oxidase of Aspergillus niger is a copper-carbonyl enzyme, which was found in the mid 1960s, purified and crystallized [l, 21. Copper and an undetermined carbonyl cofactor were found to function in the catalytic cycle [2-41. Nowadays, the importance of the fungal amine oxidase has risen again because the enzyme can be applied to monitor histamine contents in fishery products [5]. A massive production of the enzyme on the industrial scale has increased current interest.Recently, we have found two amine oxidases (EC 1.4.3.6), named AO-I and AO-11, in Aspergillus niger mycelia grown on n-butylamine medium [6, 71. AO-I is the enzyme previously reported, consisting of two 75-kDa subunits, while AO-I1 is a novel enzyme of 80 kDa. These amine oxidases do not show big differences in their peptide maps, but differ in their N-terminals. They also do not show any remarkable difference in substrate specificity or sensitivity to inhibitors. The cofactors of both enzymes give a typical reaction with p-nitrophenylhydrazine [6, 71 like other topaquiCorrespondence to I. Frkbort, Laboratory of Applied Microbiology, Faculty of Agriculture, Yarnaguchi University, 1 677-1 Yoshida, Yamaguchi, Japan 753Abbreviations. AO-I and AO-11, Aspergillus niger amine oxidases ; Nbs,, 5,5'-dithio-bis-(2-nitrobenzoic acid) ; buffer A, 0.01 M potassium phosphate pH 7.0.Enzymes. Amine oxidase, arnine:O, oxidoreductase (dearninating) (copper containing) (EC 1.4.3.6) ; peroxidase from horseradish (EC 1.11.1.7); pronase P from Streptomyces griseus (EC 3.4.24.31); thermolysin from Bacillus themolyticus rokko (EC 3.4.24.27 It is very probable that AO-I was purified and crystallized in earlier studies but fractions containing AO-I1 were not purified further because of its lower...

Towards the development of selective amine oxidase inhibitors

“…Despite the relatedness of 2 to DABY (by the elements of HCl), our finding that AGAO inactivated by DABY retains full TPQ redox competence in the Nitroblue tetrazolium assay supports a cofactor‐sparing side‐chain modification mechanism of inhibition in this case, possibly involving cyclocondensation onto a nucleophilic residue at the active site channel demonstrated by Frebort et al . for DABY inactivation of Aspergillus niger AO and grass pea AO [54]. However, inactivation of BPAO by DABY was accompanied by loss of TPQ redox competence, in agreement with what was found for 2 with both BPAO and AGAO.…”

Molecular mode of interaction of plant amine oxidase with the mechanism‐based inhibitor 2‐butyne‐1,4‐diamine

“…It has been noticed that the amine oxidase becomes brownish in colour when mixed with DABI and this is represented by remarkable changes in absorption spectrum. A dramatic increase in absorption at 295 nm which is time‐dependent, usually limiting in about 15 min, was observed as a peak at 310 nm in difference spectrum against the native enzyme from pea [2], A. niger [3] and also GPAO. The new absorption maximum can be used in a spectrophotometric titration to assess the molar ratio of the inhibitor/enzyme that is necessary for complete enzyme inactivation (Fig.…”

“…The DABI‐inactivated enzyme shows positive analytical reaction for pyrrole when derivatized with either p ‐dimethylaminobenzaldehyde [2,3] or with p ‐dimethylaminocinnamaldehyde. The absorption spectra of derivatized GPAO and derivatized pyrrole as a model compound are shown in Fig.…”

“…2‐Butyne‐1,4‐diamine (DABI) is an acetylenic substrate analogue of putrescine (butane‐1,4‐diamine) that acts as mechanism‐based inhibitor of copper amine oxidases from the Fabaceae family plants such as pea seedlings ( Pisum sativum ) with the kinetic constants r = 17, k in = 4.9 min −1 and K ′ = 0.32 m m [2] and also from fungus Aspergillus niger [3]. The proposed scheme for this catalytic reaction is shown in Eqn (1).…”

“…An active site nucleophile attacks the 4‐amino‐2‐butynal, forming covalently bound pyrrole in the active site. In the case of the A. niger amine oxidase the attacking nucleophile was determined as the amine group of Lys359 from the peptide sequence Lys‐Met‐Pro‐Asn‐Ala [3]. In this work, we focus the mechanism of enzyme inactivation using the amine oxidase from grass pea that shows high similarity to the sequence and structure of amine oxidase from pea seedlings [4,5].…”