The possible graph shapes for one-site/two-state and substrate-modifier models are discussed. The two-state model is a version of the Monod-Wyman-Changeux model and gives a rate equation with 240 denominator terms. Discussion in terms of K and V effects is not possible. A simplified version of the mechanism can be shown to give v-versus-[S] curves that are either sigmoid or non-sigmoid. They may show substrate inhibition or no final maximum, and the double-reciprocal plots can be concave up or down. The corresponding binding model is determined by only two constants and gives a linear double-reciprocal plot. The substrate-modifier mechanism is a simple example of a mechanism where inclusion of catalytic steps leads to a genuine increase in degree of the rate equation. The v-versus-[S] curve can show such complexities as two maxima and a minimum, and the double-reciprocal plot can cross its asymptote twice, proving the rate equation to be 4:4. A simplified version is 3:3, and analysis shows that at least 18 of the 27 double-reciprocal plots that can arise with 3:3 functions are possible with this particular mechanism. Representative double-reciprocal and Scatchard plots are presented for several sets of rate-constant values. It is concluded that relatively simple mechanisms give pseudo-steady-state rate equations of high degree and considerable complexity. With extended ranges of substrate concentrations there is every reason to believe that experimental data would show the sort of deviations from Michaelis-Menten kinetics seen with calculated curves for such simple mechanisms. Narrow ranges of substrate concentration, on the other hand, would lead to inflexions and curvature being overlooked. It is not possible to discuss such deviations from Michaelis-Menten kinetics in terms of kinetic constants such as Km and V, and, in general, it is also difficult to see any simple way to explain intuitively such features as sigmoidicity, substrate inhibition, double-reciprocal convexity and decrease in degree by cancellation of common factors between numerator and denominator of rate equations. These conclusions apply with even more force when catalytic steps are included, for then the rate equations, are for multi-site mechanisms, of higher degree, allowing increasingly complex curve shapes. A number of enzymes were studied and initial-rate data were fitted by computer.(ABSTRACT TRUNCATED AT 400 WORDS)
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.
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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