Amino acid oxidase reaction

Amino acids (isoleucine, phenylalanine, arginine and alanine) have been analysed on a microchip with a post-channel reaction with amino acid oxidase reaction [144], Pre-channel derivatisation of amino acids with naphthalene-2,3-dicarboxyaldehyde (NDA) has been described for facilitating its amperometric detection [145]. Separation and direct detection of amino acids without derivatisation have also been achieved in microchips [89,109,122,132,146-148]. 

Porter, D. J. T., Voet, J. G., and Bright, H. J., 1977, Mechanistic features of the D-amino acid oxidase reaction studied by double stopped flow spectrophotometry, J. Biol. Chem. 252 4464n4473. 

When catalase activity is absent, H2O2 can be monitored directly spectrophotometrically (29) or by coupling to a peroxidase system (30). The weak n tt transition of the carbonyl group is present in the oc-keto acid products of the amino acid oxidase reactions and can also be utilized under special conditions (31), However, all these methods present far more serious technical problems than the O2 electrode technique and are used only in special circumstances. 

D-Amino Acid Oxidase. The kinetic mechanism of this reaction is qualitatively, and quantitatively to some extent, very similar to that for the L-amino acid oxidase reaction. The long-wavelength intermediate (Er P) is generally much more obvious in this reaction because of the remarkable slowness of P liberation. Yagi (3) has crystallized this intermediate under anaerobic conditions. Since is often, with different substrates, many orders of magnitude smaller than the maximum turnover... 

The intermediate EiP, which is the major species of reduced enzyme with which O2 reacts in the amino acid oxidase reaction, is more reactive with O2 than Er in one case (49) (D-amino acid oxidase) but less reactive in the other (18) (n-amino acid oxidase). The reasons for such seemingly inconsistent behavior, as well as the virtual lack of reactivity of reduced flavins with O2 in systems such as succinic dehydrogenase, will only become clear when the molecular details of the oxidation mechanism of reduced flavin are elucidated. 

Until recently, the amino acid oxidase reaction has been studied only in the direction of ammonia and a-keto acid formation. In the presence of air the reaction proceeds to completion and is essentially irreversible because of the reoxidation of the reduced flavoprotein by molecular oxygen [reaction (5)]. Meister and his associates 11, 12) have provided a clear demonstration of the reversibility of the amino acid oxidase reaction with D-amino acid oxidase (from sheep kidney) and L-amino acid oxidase (from snake venom). When an amino acid, ammonia, and the a-keto acid analog of a second amino acid are incubated with either amino acid oxidase under anaerobic conditions, the formation of the second amino acid is observed ... 

The reaction is markedly accelerated by the addition of ammonia leads to the formation of the N Mabeled amino acid and spectrophoto-metric studies show that the reduced amino acid oxidase can be reoxidized anaerobically by the addition of ammonia and a-keto acid. All oi these experiments indicate that the reaction observed does not involve a transamination. It has been suggested that imder appropriate phyinological conditions a reversal of the amino acid oxidase reaction may be responsible for amino acid synthesis. 

Flavin oxidation of carbanions has also been of much concern since active intermediates in some flavoenzyme-mediated reactions (amino acid oxidase, lactate oxidase, etc.) are carbanions (Kosman, 1977). Flavin oxidation of nitroethane carbanion (20), which had not been achieved in non-enzymatic systems, occurs with [56] bound to CTAB micelles (Shinkai etal., 1976b). This suggests that the nitroethane carbanion is also activated by the micellar environment. 

FIGURE 4.19 Amino acid enantiomers are determined by reaction (A) with l- or D-amino-acid oxidase at pH 7-8.75 Added catalase decomposes the hydrogen peroxide (B), which would otherwise oxidize the a-oxoacid. Quantitation is achieved by measuring oxygen consumption, which is 0.5 mol/mol of substrate. 

In order to follow the progress of an enzyme-catalysed reaction it is necessary to measure either the depletion of the substrate or the accumulation of the product. This demands that either the substrate or the product show some measurable characteristic which is proportional to its concentration. This is not always the case and a variety of techniques have been developed in order to monitor enzyme reactions. In order to illustrate some of the methods and also to give an appreciation of the technical details and the calculations, three examples are given (Procedures 8.4 to 8.6) that use the enzyme D-amino acid oxidase (Table 8.4). 

Figure 8.14 The effective analytical range of an enzyme assay. The assay of D-amino acid oxidase (EC 1.4.3.3), using the method detailed in Procedure 8.5, shows a valid analytical range up to a maximum reaction rate of 0.10 absorbance change per minute.

Test reaction — catalysed by D-amino acid oxidase... 

L-Amino acid oxidase has been used to measure L-phenylalanine and involves the addition of a sodium arsenate-borate buffer, which promotes the conversion of the oxidation product, phenylpyruvic acid, to its enol form, which then forms a borate complex having an absorption maximum at 308 nm. Tyrosine and tryptophan react similarly but their enol-borate complexes have different absorption maxima at 330 and 350 nm respectively. Thus by taking absorbance readings at these wavelengths the specificity of the assay is improved. The assay for L-alanine may also be made almost completely specific by converting the L-pyruvate formed in the oxidation reaction to L-lactate by the addition of lactate dehydrogenase (EC 1.1.1.27) and monitoring the oxidation of NADH at 340 nm. 

Fig. 5.23. Mechanism of oxidative opening of azaheterocycles. Hydroxylation at the a-posi-tion (Reaction a) yields an unstable carbinolamine, which is in equilibrium with an open-chain amino aldehyde. The carbinolamine can be converted by aldehyde oxidase to a lactam derivative (Reaction b), while the open-chain amino aldehyde can be converted by aldehyde dehydrogenase to a ft)-amino acid derivative (Reaction c). 

Reduction of D-proline by D-amino acid oxidase at pH 8 shows two steps when monitored at 640 nm. These are interpreted as the build-up and breakdown of a reduced enzyme-imino acid charge transfer complex. If the reaction is monitored using phenol red the same two rates are observed but additionally the release of = 1 proton for each step can be assessed and interpreted. The indicator changes are followed at 505 nm and 385 nm, which are isosbestic wavelengths for the two steps (without indicator),... 

Figure 8.6 The three dehydrogenase (oxidase) reactions in amino acid degradation. The enzymes are D-amino acid oxidase, glutamate dehydrogenase and proline oxidase (dehydrogenase). Biochemical details are given in Appendix 8.4.

This FMN-dependent enzyme [EC 1.1.3.15], also known as (5)-2-hydroxy-acid oxidase, catalyzes the reaction of a (5)-2-hydroxy acid with dioxygen to produce a 2-oxo acid and hydrogen peroxide. The enzyme exists as two major isoenzymes. The A form of the protein preferentially oxidizes short-chain aliphatic hydroxy acids. The B form preferentially oxidizes long-chain and aromatic hydroxy acids. The rat isoenzyme B form also acts as an L-amino-acid oxidase. 

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