N-Amino acid oxidases

Glucose oxidase is obtained from molds such as Aspergillus niger and species of Penicillium, In its gross molecular properties at least, the enzyme from A. niger resembles l- rather than n-amino acid oxidase (19) since it is a dimer of 186,000 MW with two tightly, but noncovalently, bound FAD molecules per dimer. These FAD moieties are kinetically independent and probably, therefore, are present at an active site on each subunit. The enzyme is remarkably stable over a wide range of pH and other variables, which makes it ideal for analytical and bulk conversion applications. 

The expected result was obtained since n-amino acid oxidase converted )8-chloroalanine to pyruvate under anaerobic conditions, to chloropyruvate at high O2 concentrations, and to mixtures of these at intermediate O2 concentrations. Under steady state conditions, the reaction behaved as if cleavage of the a C-H bond were the rate-limiting process in turnover, although stopped-flow spectrophotometric measurements showed that this interpretation can not be entirely correct in this case (53) or in the case of )8-choloro-a-aminobutyrate (54), a-yS-Elimination has now been observed in three flavoprotein oxidase reactions (54) and can be considered strong circumstantial evidence for a-proton removal from compounds which closely resemble the physiological substrates. 

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. 

Stadie, W. C., and Haugaard, N., Oxygen poisoning. VII. The effect of high oxygen pressure upon enzymes uricase, xanthine oxidase, and n-amino acid oxidase. J. Biol. Chem. 161, 181-188 (1945). 

The above hypothetical mechanism is, of course, based on the view that a-iminoglutarate is indeed an intermediate in the reaction. It has been shown that a-imino acids are intermediates in the reactions catalyzed by the flavin-containing l- and n-amino acid oxidases (328,3 7). In addition, such a mechanism would be consistent with the observed difference in pH optima for the forward and reverse reactions (Table... 

Oxidases use oxygen as electron acceptor and transfer two or four electrons. Those which transfer two electrons form H2O2. They include Glucose oxidase (see), the iron- and molybdenum-containing Xanthine oxidases (see), and the n-amino acid oxidases (containing FMN or FAD). The latter catalyse the irreversible formation of the corresponding a-ke-toacid. Oxidases which transfer four electrons contain copper they oxidize the substrate and form water, e.g. laccase, ascorbate oxidase and p-diphenol oxidase. 

This is the flavoprotein glucose oxidase, and the hydrogen peroxide produced is coupled to the formation of a green d[ye using the colourless leuco-dye 2,2 -azino-di-(3-ethylbenzthiazoline) 6-sulphonate [ABTS], This simple format can be used with other enzymes that are specific for the substrate they oxidize and produce hydrogen peroxide as a product (for example, galactose oxidase, n-amino acid oxidase, monoamine oxidase etc.). 

The reaction carried out by n-amino acid oxidase uses molecular oxygen as electron acceptor efficiently. The conventional assay of this... 

Simple Flavoproteins. The specificity of kidney n-amino acid oxidase for n-amino acids is discussed in Chapter VII. Similar flavoproteins have... 

Although D-amino acids have recently been found in a number of naturally occurring materials, the total amounts are modest, and the physiological role of n-amino acid oxidase remains obscure. 

L-Amino acid oxidase of rat kidney appears to catalyze the same type of reaction as n-amino acid oxidase. It oxidizes the monoamino, mono-carboxylic acids, but does not attack either dicarboxylic or polybasic compounds. Proline and W-methylamino adds are oxidized. A unique property of this enzyme is its ability to oxidize hydroxy acids. Only L-hydroxy acids are attacked. The ratio of hydroxy acid oxidation rate to amino acid rate is constant throughout purification. In general, hydroxy acid oxidation proceeds somewhat more rapidly than amino add oxidation, and the hydroxy acids corresponding to the basic amino adds... 

Ti-Aspartic Oxidase. Aspartase and transaminases account for a major part of the metabolism of L-aspartic acid. n-Aspartic acid is oxidized by an enzyme present in liver and kidney. This is an oxidase that converts aspartate to oxalacetate and ammonia while reducing oxygen to hydrogen peroxide. The oxidase was resolved by ammonium sulfate precipitation and dialysis to a protein that could be reactivated by FAD but not by FMN. The enzyme differs from n-amino acid oxidase in its insensitivity to benzoate. The only other known substrate for the partially purified D-aspartic oxidase is D-glutamate, but since the relative rates of oxidation of the two amino acids vary during the preparation of the enzyme, it is... 

This classification indicates that the L- and n-amino acid oxidases have broad substrate specificities, w hereas the other enzymes have a narrower specificity. For the purpose of simplicity the above classification will be used. The amino acid oxidase systems may be subclassified according to the nature of the hydrogen acceptor under two categories, aerobic and anaerobic. The former have been more commonly referred to as amino acid oxidases, whereas the latter are frequently referred to as dehydrogenases. 

Keilin and Hartree demonstrated the coupling of n-amino acid oxidase with ethyl alcohol in the presence of catalase and were able to show that in the presence of catalase ethyl alcohol was oxidized in preference to the a-keto acid to form acetaldehyde as follows ... 

Lange, JJd., Venhorst, J., van Dongen, M.J. et al (2011) Biophysical and physicochemical methods differentiate highly ligand-effident human n-amino acid oxidase inhibitors. European Jownal of Medical Chemistry, 46, 4808-4819. 

D- and L-leucine have been prepared by amination of n- and u-o-bromoisocaproic acid (296) and by the action of pancreatin on oi/-leudne (846). t/-Leudne has been prepared by the action of P, gUrucim on Di/-leudne (702, 704). n-Leudne has been prepared by the action of n-amino acid oxidase on DL-leudne (766). 

As indicated above, the reaction catalyzed by the general d- and l-amino acid oxidases has been represented by a dehydrogenation of an amino acid by a flavoenzyme to jneld reduced flavoenzyme and the corresponding imino acid [reactions (2) - - (4)]. Indirect support for the formation of the hypothetical imino acid has been provided by a number of studies which exclude a,/3-unsaturation in the course of the reaction. For example, it has been shown that (a) the four isomers of isoleucine are enzymically oxidized by the appropriate amino acid oxidase to the corresponding optically active a-keto-/3-methylvaleric acids (5, 6), (b) the l-isomers of /3-phenylserine are converted by L-amino acid oxidase to the respective isomers of mandelic acid (7), (c) the l- and D-isomers of a-aminophenylacetic acid, which have no /3-hydrogen atom, are attacked by the amino acid oxidases 8, 9), aod (d) on the oxidation of L-leucine in the presence of D,0 by L-amino acid oxidase, no deuterium is found in the isolated a-ketoisocaproic acid (10). More direct evidence for the formation of the a-imino acid as an intermediate has been provided by Pitt (10a) in studies on the oxidation of aromatic amino aci by ophto-n-amino acid oxidase in the presence of a tautomerase. 

Type of amino acid n-Amino acid oxidase of sheep kidney L-Amino acid oxidase of cobra venom L-Amine acid oxidase of N. crAmino acid oxidase of N. cra a L-Amino acid oxidase of Mytiba eduH ... 

The second type of inhibition, i.e., competitive inhibitors for the amino acid substrates, is that produced by aromatic compounds. Benzoate is a remarkably potent competitive inhibitor of mammalian n-amino acid oxidase (6S, 64)- A detailed study of benzoic acid derivatives as competitive inhibitors has been carried out by Bartlett (64)- The structural unit necessary for inhibition is the phenylcarboxylate ion. Ring substitution has a less marked efiFect, although in general meta substitution, particularly by a halogen, methyl, or nitro group, results in greater inhibitory activity than does substitution in other positions. Benzoate competes with the natural substrates for the holoenzyme and has little or no effect on the apoenzyme. Its inhibitory action is reversible (6S, 65). Heterocyclic acids with aromatic properties (66) are also inhibitors. 

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