D-Amino acid oxidase, reaction

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. 

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... 

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.

A modification of this reaction concerns the availability of the keto acid substrate. To circumvent its complicated lengthy chemical synthesis, 2-keto-6-hydro-xyhexanoic acid was synthesized by treatment of racemic 6-hydroxynorleucine with D-amino acid oxidase and catalase (Fig. 37). The production of racemic 6-hydroxynorleucine occurs by hydrolysis from 5-(4-hydroxybutyl)hydantoin. d-Amino acid oxidase converts the D-enantiomer of racemic 6-hydroxynorleucine to the corresponding ketoacid which is reductively aminated to l 6-hydroxynorleucine by GluDH. 

A combination of D-amino acid oxidase and L-amino transferase is an example of a deracemization by stereoinversion. The product is an L-amino acid. The reaction catalyzed by amino transferase has an equilibrium constant close to unity, a very unpractical situation leading to uncomplete transformation and to the production of almost inseparable mixtures of amino acids (at least two, the amino acid product and the amino add used as an amino donor). For preparative purposes it is therefore mandatory to shift the equihbrium to the product side. A recent example of a deracemization procedure based on this coupled enzymatic system is the preparation of L-2-naphthyl-alanine 6 as illustrated in Scheme 13.9 [28]. The reaction occurs in one pot with initial oxidation of the D-amino acid catalyzed by D-amino acid oxidase from Rhodotonda gracilis. The hydrogen peroxide that is formed in stoichiometric amounts is decomposed by catalase. The a-keto add is the substrate for L-aspartate amino transferase (L-Asp amino transferase), which is able to use L-cysteine sulfinic acid 7 as an amino donor. 

Denu, J. M., and Fitzpatrick, P. F., 1994, Intrinsic primary, secondary, and solvent kinetic isotope effects on the reductive half-reaction of D-amino acid oxidase evidence against a concerted mechanism. Biochemistry 33 400194007. 

D-Amino acids can be determined in the presence of L-amino acids with D-amino acid oxidase, which is found in the kidney and liver of all animals, particularly the sheep and pig. In this reaction, the D-amino acid is converted to an a-keto acid, ammonia, and hydrogen peroxide, with the uptake of O2 (Equation 15). 

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... 

For these reasons, progress has been obtained with model, rather than physiological, substrates. In particular, recent studies of the reaction of the amino acid oxidases with )8-halogenated-a-amino acids and of D-amino acid oxidase and glucose oxidase with nitroalkanes and their carbanions have begun to clarify the chemical mechanism of these reactions. The results and interpretations of these studies are discussed briefly below. 

Studies of nitroalkane oxidation by n-amino acid oxidase (55) and glucose oxidase 49, 56) have provided strong evidence both for intermediate substrate carbanions and for subsequent covalent adduct formation between these and the N position of the flavin nucleus. The rationale for using nitroalkanes can be seen in the following reaction stoichiometries for D-amino acid oxidase (55) ... 

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. 

---