Benzylamine oxidase from pig plasma has been studied by a variety of chemical and physical techniques. 1. Analytical ultracentrifugation, gel electrophoresis and isoelectric-focusing studies suggest that the enzyme is composed of two subunits with closely similar primary structures. 2. E.s.r. and n.m.r. measurements show that the enzyme contains two well-separated (greater than 0.6 nm) Cu2+ ions at chemically distinct sites. Each Cu2+ ion is coordinated by two water molecules, one 'axial' and the other 'equatorial'. Both water molecules undergo fast exchange (10(5)--10(8) s-1) with solvent and are deprotonated in the pH range 8--9, but only the equatorial water molecule is displaced by the inhibitors N3- and CN-. 3. Kinetic and e.s.r. measurements show that azide and cyanide compete against O2 binding and also make the two Cu2+ sites identical. It is concluded that Cu2+ must participate in the re-oxidation of reduced enzyme by molecular O2.

1. Isoelectric focusing studies on human placental diamine oxidase showed the pl value of the active enzyme to be 6.5. This infonnation was used in modifying the enzyme purification by incorporating column chromatography on DEAE-Sephadex with ionic strength and pH-gradient elution and this, together with affinity chromatography on concanavalin A-Sepharose, gave a highly purified preparation, with a specific activity of 7.0 units/mg. 2. The enzyme gave the expected stoicheiometry with p-dimethylaminomethylbenzylamine as substrate (Keq. 2700) and also oxidized [8-arginine]vasopressin, [8-lysine]vasopressin, collagen and tropocollagen. Polyacrylamide gel slices showed identical migration ofdiamine-oxidizing and [8-lysine]vasopressin-oxidizing activity. 3. The molecular weight, determined by ultrantrifugation, sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, variable polyacrylamide-gel electrophoresis and Sephadex G-200 column chromatography, was estimated to be approx. 70000. 4. E.s.r. spectroscopy showed that copper and manganese were present in the purified enzyme. This result was confirmed by atomic absorption spectroscopy, which indicated a stoicheiometry for copper and manganese of approx.

Protection against inhibition of creatine kinase by iodoacetamide is measured by the decrease in the rate constant for the inhibition reaction; A mixture of purified substrates at equilibrium protects quite strongly when all the components of the mixture are nearly saturating. The protection by substrates 'working' in the forward direction only (from creatine and MgATP) was measured by carrying out the experiment rapidly at low concentrations of the enzyme; by varying the concentration of substrate it was found that the amount of protection when the substrates of the forward reaction are saturating is about 80% (100% protection would imply a value of zero for the rate constant of the inhibition reaction). The effects of Ca2+ and Mg2+ are compared. It is already known that the complex creatine-NO3--MgADP, which is considered to be either a transition-state analogue or an analogue of an intermediate in the reaction pathway, protects fully against iodoacetamide, whereas creatine and MgADP alone, or together without NO3-, do not protect. This suggests that the degree of protection by the working enzyme represents the proportion of enzyme molecules that have a conformation complementary to a creatine-PO3-MgADP intermediate.

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...

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