O-Amino acid oxidase

In the Penzyme test, freeze-dried Streptomyces oo-carboxypeptidase is placed in sealed vials to which the milk sample is added. A preliminary incubation is carried out for 5 min at 47 C to cause some inactivation of the enzyme molecules. The degree of inactivation is dependent on the amount of -lactams presumed to be present in the milk sample. Subsequent addition of a reagent tablet containing synthetic o-alanine oligopeptide and o-amino acid oxidase followed by reincubation at 47 C for 15 min results in the release of o-alanine, its amount... 

Immobilized o-amino acid oxidase from T.variabilis... 

A long-known characteristic of o-amino acid oxidase is its tendency to form charge-transfer complexes with amines, complexes in which a nonbonding electron has been transferred partially to the flavin. Complete electron transfer would yield a flavin radical and a substrate radical which could be intermediates in a free radical mechanism, as discussed in the next section. ... 

To understand the carbanion mechanism in flavocytochrome 62 it is useful to first consider work carried out on related flavoenzymes. An investigation into o-amino acid oxidase by Walsh et al. 107), revealed that pyruvate was produced as a by-product of the oxidation of )8-chloroalanine to chloropyruvate. This observation was interpreted as evidence for a mechanism in which the initial step was C -H abstraction to form a carbanion intermediate. This intermediate would then be oxidized to form chloropyruvate or would undergo halogen elimination to form an enamine with subsequent ketonization to yield pyruvate. The analogous reaction of lactate oxidase with jS-chlorolactate gave similar results 108) and it was proposed that these flavoenzymes worked by a common mechanism. Further evidence consistent with these proposals was obtained by inactivation studies of flavin oxidases with acetylenic substrates, wherein the carbanion intermediate can lead to an allenic carbanion, which can then form a stable covalent adduct with the flavin group 109). Finally, it was noted that preformed nitroalkane carbanions, such as ethane nitronate, acted as substrates of D-amino acid oxidase 110). Thus three lines of experimental evidence were consistent with a carbanion mechanism in flavoenzymes such as D-amino acid oxidase. 

Scheme 3 Proposed reaction mechanisms of /3-chioroaianine with o-amino acid oxidase.

Scheme 4 Alternative mechanisms of amino acid oxidation by o-amino acid oxidase.

There are several examples of d to l inversion of amino acids in the literature. D-Phenylalanine may have therapeutic properties in endogenous depression and is converted to L-phenylalanine in humans [145]. o-Leucine is inverted to the L-enantiomer in rats. When o-enantiomer is administered, about 30% of the enantiomer is converted to the L-enantiomer with a measurable inversion from l to o-enantiomer. As indicated in Fig. 13, D-leucine is inverted to the L-enantiomer by two steps. It is first oxidized to a-ketoisocarproate (KIC) by o-amino acid oxidase. This a-keto acid is then asymmetrically reaminated by transaminase to form L-leucine. In addition, KIC may be decarboxylated by branched-chain a-keto acid dehydrogenase, resulting in an irreversible loss of leucine (Fig. 13) [146]. D-Valine undergoes a similar two-step inversion process, and this can be antagonized by other amino acids such as o-leucine. The primary factor appears to be interference with the deamination process [147]. 

A final distinction from nicotinamides is that the flavin coenzymes generally form tight non-dissociable non-covalent complexes with the apoenzyme. Nicotinamides are released at the end of each catalytic cycle and so are consumed as substrate as part of the redox stoichiometry. Because flavins are tightly bound to the apoprotein 10 -10 " M) the coenzyme must be oxidised/reduced at the end each turnover before the enzyme complex again becomes catalytically active. Differential binding of flavin and dihydroflavin is responsible for the wide range of redox potentials for flavoproteins so that oxidation or reduction can be thermodynamically favourable. For example, o-amino acid oxidase binds FAD with a dissociation constant of 10 M but FADHj with one of 10"M which changes the reduction potential from —200 for the FAD/FADHj couple free in solution to 0 mV when bound to the enzyme. 

P. are particularly abundant in liver and kidney cells, e.g. a single rat liver cell contains 350-400 P. The half-life of P. is 1.5-2 days. Other cell types usually contain smaller P. with homogeneous contents (microperoxisomes). In addition to catalase, P. contain, inter alia, o-amino acid oxidase, a-hydroxyacid oxidase and urate oxidase. The latter enzyme is often present as a large crystal in the otherwise homogeneous matrix. These enzymes are particularly important in the oxidative degradation of metabolic intermediates (e.g. purine bases) and in the formation of carbohydrates from amino acids and other materials. [L.J. Olsen J.J.Harada Peroxisomes and Their Assembly in Higher Plants Annu. Rev. Plant Physiol. Plant Mol. BioL 46 (1995) 123-146)... 

Fujii T, Yamamoto K, Yamamoto S, Matsumoco K, Mizuno M. Cephalosporin compound preparation—by reaction of cephalosporin C with o-amino acid oxidase producing fbngus. Jpn Kokai Tokkyo Koho (Jpn Patent Application) JP 51044695 1976. 

D-Aspariic Acid Oxidase. Still et al. reported that rabbit kidney and liver contain a soluble enzyme which catalyzes the aerobic oxidation of D-aspartate to oxalacetate plus NH3 with the formation of hydrogen peroxide. In a later study by Still and Sperling the D-aspartic acid oxidase was resolved and reactivated by the addition of FAD. The purified enzyme showed about one-sixth the activity with D-glutamate this, according to these workers, is best explained by the presence of a D-glu-tamic acid oxidase. The activity of n-aspartic acid oxidase is higher than that of D-amino acid oxidase in rabbit kidney and liver, and they are of the same order of activity in pig kidney. In contrast to pig kidney o-amino acid oxidase, which is inhibited by benzoic acid, the D-aspartic acid oxidase was unaffected. 

Chemists can use an enzyme s ability to distinguish between enantiomers to separate them. For example, the enzyme o-amino acid oxidase catalyzes only the oxidation of the R enantiomer but leaves the S enantiomer unchanged. The oxidized product of the enzyme-catalyzed reaction can be easily separated from the unreacted enantiomer because they are different compounds. 

Because the resolution (separation) of the enantiomers depends on the difference in the rates of reaction of the enzyme with the two A -acetylated compounds, this technique is known as a kinetic resoiution. In Section 6.17, we saw that a racemic mixture of amino acids can also be separated by the enzyme o-amino acid oxidase. 

Although D-amino adds can no longer be considered unnatural, thoy are, from a quantitative standpoint, neverthdess, of limited distribution in nature. This fact makes the explanation of the widespread occurrence of hi y active o-amino acid oxidases somewhat difficult. The capriciousness of nature—or of the enzymologist—is clearly revealed in the fact that a great deal is known about the properties and mechanism of action of D-amino add oxidases in the animal but very little about the role of D-amino acids in metabolism. 

In 1964, Yahiro et al. demonstrated enzyme mediation to platinum electrodes [21], They used three enzymes GOx, D-amino acid oxidase, and alcohol dehydrogenase (ADH). If iron was introduced into the system with either GOx or o-amino acid oxidase, an electrical potential was observed. However, this was not the case with ADH, which is dependent on a nicotinamide adenine dinucleotide (NAD) cofactor (see below). 

Structure of the Mutant Porcine Kidney o-Amino Acid Oxidase (Y228L, R283G)... 

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