Steady‐State Kinetic Studies on Benzylamine Oxidase from Pig Plasma

Abstract

Steady-state kinetic studies on the enzyme benzylamine oxidase from pig plasma are described. Eadie-Hofstee plots with benzylamine as the varying substrate are non-linear; examination of this data indicates that the observed effects are probably due to the amine substrate participating in at least two reactions with enzyme. Ammonia and imidazole modify the activity of the enzyme; under specified conditions of pH or modifier concentration, the effect on the activity can be either activation or inhibition. Eadie-Hofstee plots of the data establish that the modifier also participates in at least three reactions with the enzyme. Eadie-Hofstee plots at pH 9 with oxygen as the varying substrate are linear, which allows kinetic parameters to be determined. From studies on the effect of ammonia and imidazole on these parameters, information has been derived on how these modifiers affect component steps of the catalytic cycle.Recent studies on benzylamine oxidase from pig plasma have clarified some aspects of the catalytic mechanism [1,2]. The enzyme molecule, composed of two subunits, has two tightly bound cupric ions [I] and one catalytically-active carbonyl grouping [2]. Magnetic resonance methods have provided information on the ligands coordinated to the copper and have shown also that the two cupric ions are non-identical [l]. Only one of the cupric ions appears to be modified during the catalytic cycle [3], an observation which is consistent with there being a single active site [2,4]. The extensive stopped-flow kinetic studies made by Pettersson and co-workers [5] have led to the minimal mechanism shown in Scheme 1. This scheme suggests that the enzyme binds only once with its amine substrate during each catalytic cycle. However, steady-state kinetic studies revealed curved double-reciprocal plots under certain conditions which points to the mechanism of Scheme 1 being oversimplified [ 6 ] ; the mechanistic significance of the curved double-reciprocal plots was not discussed in this paper.The aims of the present investigation were to assess the complexity of the mechanism by examining in more detail the manner in which amine substrate and other effector molecules modify enzyme activity. It is shown that both amine substrate and effectors participate in more than one reaction with the enzyme; the data further allow three sites of action of the modifiers in the catalytic cycle to be identified. MATERIALS AND METHODSBenzylamine oxidase was purified as described previously [6] and stored at 4°C as a crystalline suspension in amEnzyme. Benzylamine oxidase or amine : oxygen oxidoreductase (deaminating) (EC 1.4.3.6). monium sulphate solution. All chemicals used were of reagent quality and were purchased from British Drug Houses (Poole, Dorset, UK). Benzylamine and p-methylbenzylamine were converted to the hydrochloride salts and re-crystallised twice from aqueous ethanol before use whilst imidazole was re-crystallised from acetone. Enzyme for the kinetic studies was dialysed at 4°C against two changes (each of 1000 vol...

Identification of Two Imidazole Binding Sites and Key Residues for Substrate Specificity in Human Primary Amine Oxidase AOC3

“…The shallowness of the Hill slope (Figure ) suggests that imidazole inhibits hAOC3 in a rapidly reversible manner, and we were able to demonstrate almost uninhibited activity of hAOC3-expressing CHO cells after removal of imidazole (data not shown). The inhibitory effect of a high imidazole concentration has actually been reported long ago for pig plasma amine oxidase . More recently, efficient inhibition and covalent binding of secondary amines to TPQ have been reported in BSAO .…”

Allosteric modulation of semicarbazide‐sensitive amine oxidase activities in vitro by imidazoline receptor ligands

“…Activation of SSAO enzymes in vitro is not entirely without precedent. Elegant kinetic work with porcine plasma amine oxidase by Kelly et al . (1981) revealed that this enzyme could be activated and inhibited by ammonia, a product of oxidation of primary amines by SSAO, and by imidazole, a common constituent in biological buffers.…”

“…While lysophosphatidylcholine was shown also to activate lung SSAO, the efficacy of this plasma phospholipid was rather lower than that of plasma itself, and it was suggested that other components in the plasma may be of greater importance in this regard ( Dalfó et al ., 2003 ). Nevertheless, the demonstration that a tissue‐bound SSAO enzyme can be activated in vitro through effects of an endogenous species on substrate affinity in a manner analogous to the present observations with soluble SSAO enzymes, and to those of others ( Kelly et al ., 1981 ), suggests that allosteric sites similar to those identified in the present study may be present on several different amine oxidase enzymes, and may be physiologically relevant. Furthermore, the findings here and by others of SSAO activation in enzymes from several species and tissue sources, and in both impure and purified enzyme preparations, argue against the likelihood that the imidazoline drugs bind to a second contaminating protein and that this protein–drug complex then interacts with the SSAO dimer to activate or inhibit substrate turnover.…”

“…Elegant kinetic work with porcine plasma amine oxidase by Kelly et al (1981) revealed that this enzyme could be activated and inhibited by ammonia, a product of oxidation 504 A. Holt et al Modulation of amine oxidases by imidazolines of primary amines by SSAO, and by imidazole, a common constituent in biological buffers. Furthermore, the authors concluded that the simplest model that might explain their observations would possess two sites, in addition to the active site, at which modifiers might bind (Kelly et al, 1981). More recently, a component of human plasma was found to activate SSAO from human lung through increasing affinity for benzylamine substrate (Dalfo´et al, 2003).…”

“…This effect was not commented upon by the authors. A related amine oxidase from porcine plasma was stimulated in vitro , in a pH‐dependent manner, by including ammonia or imidazole in the enzyme reaction ( Kelly et al ., 1981 ; Yadav & Knowles, 1981 ).…”