D-Amino Acid Oxidase EC

The most investigated D-amino acid oxidases are those from the yeasts R. gracilis and T. variabilis and the one from pig kidney. In solution D-amino acid oxidases are homodimers, with each monomer constituted by -350-370 residues and containing one molecule of non-covalently bound FAD co-factor. Mammalian and 

Protein engineering studies have allowed modulation of D-amino acid oxidases oligomerization state, stability (which is significantly increased in the immobihzed form), FAD binding, and substrate specificity (for a review see [57]). The residue 

L-Amino acid oxidase is a flavoenzyme that catalyzes the oxidative deamination of L-amino adds. L-Amino acid oxidase activities have been detected in mammals, birds, reptiles, invertebrates, molds, and bacteria [54]. L-Amino acid oxidases show the typical absorption spectrum due to the presence of a molecule of non-covalently bound FAD per subunit (with maxima at 465 and 380nm) they behave like flavoprotein oxidases, as in the case of D-amino acid oxidase. L-Amino add oxidase isolated from rat liver was reported to utilize flavin mononudeotide (FMN) as a co-enzyme, but since it is more active on L-hydroxy acids than on amino adds, it was thus considered as an L-hydroxy add oxidase. Even a partially purified L-amino acid oxidase from turkey Uver appeared to have FMN as a co-factor. 

The best substrates for ophidian L-amino acid oxidases are aromatic or, most generally, hydrophobic amino acids, polar and basic amino acids being deami-nated at much lower rates Glu, Asp, and Pro are not oxidized by L-amino add oxidase. L-Amino add oxidase is also active on ring-substituted aromatic amino acids, as well as on seleno cysteinyl derivatives. The substrate specificity depends on the source of the enzyme (e.g. Ophiophagus hannah L-amino acid oxidase also oxidizes Lys and Om) and on the pH. The of the reaction of Crotalus adaman- 

L-Amino acid oxidase undergoes two types of reversible inactivation one obtained by raising the pH from 5.5 to 7.5 and the temperature from 25 to 38 °C, while the second one is caused by storage at -5 to -60 °C and depends on the pH (it is favored by acidic pH values) and on the ionic composition of the storage buffer. In both cases reactivation is achieved by incubating the enzyme at pH 5 and 38 °C for Ih. Snake venom L-amino acid oxidases should be maintained in the dark at 4°C and near neutral pH to avoid inactivation. 

---

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.

D-Amino acid oxidase (EC 1.4.3.3) extracted from sheep kidney possesses low selectivity and at pH 8-9 will oxidise many D amino acids, whereas L-amino acid oxidase (EC 1.4.3.2) from snake venom (Crotalus adaman-teus) at pH 8-9 catalyses the oxidation of many L amino acids. However, as these enzymes show different reactivity towards different amino acids, the results for a sample that contains several D and L amino acids may be difficult to interpret. The use of these enzymes is therefore only recommended for the measurement of one isomer of an isolated amino acid. They may also be used to remove an unwanted isomer from a sample containing both to allow subsequent measurement of the other. 

Two enzymes with a broad substrate specificity have been utilized in biosensors for amino acids L-amino acid oxidase (EC 1.4.3.2) and D-amino acid oxidase (EC 1.4.3.3). They catalyze the irreversible formation of the respective a-keto acids ... 

D-Amino acid oxidase (EC 1.4.3.3) (Eq. (11)), converting glycine to glyoxylate, is present in the CNS (Gaunt and De Duve, 1976), but glycine is a very poor substrate for the enzyme (DeMarchi and Johnston, 1969). 

The enzyme aspartate decarboxylase (EC 4.1.1.11) will decarboxylate aminomalonic acid in H20 to yield (25)-[2- HJglycine and will also transaminate glyoxylic acid in H20 to yield (2K)-[2- H]]glycine (78). The chirality of the product was assayed using the pro-S specific D-amino acid oxidase (EC 1.4.3.3). 

All amino acids except glycine exist in these two different isomeric forms but only the L isomers of the a-amino acids are found in proteins, although many D amino acids do occur naturally, for example in certain bacterial cell walls and polypeptide antibiotics. It is difficult to differentiate between the D and the L isomers by chemical methods and when it is necessary to resolve a racemic mixture, an isomer-specific enzyme provides a convenient way to degrade the unwanted isomer, leaving the other isomer intact. Similarly in a particular sample, one isomer may be determined in the presence of the other using an enzyme with a specificity for the isomer under investigation. The other isomer present will not act as a substrate for the enzyme and no enzymic activity will be demonstrated. The enzyme L-amino acid oxidase (EC 1.4.3.2), for example, is an enzyme that shows activity only with L amino acids and will not react with the D amino acids. 

Tryptophan 331 is converted to tryptamine 332 by both aromatic L-amino acid decarboxylase (EC 4.1.1.28) and tyrosine decarboxylase (EC 4.1.1.25), and in both instances (334, 335) it was shown, either by use of the pro-R specific monoamine oxidase (335) or by degradation of the labeled tryptamines to glycine and use of the pro-S specific D-amino acid oxidase and pro-R specific glutamate pyruvate transaminase (334), that decarboxylation involved retention of configuration. Hydroxylation that leads to sporidesmin 333 has been shown to involve specific loss of the 3-pro-R hydrogen, and so again hydroxylation involves retention of configuration (102). 

Aerobic P. d. The amino groups of adenine and guanine are removed hydrolytically by specific deaminases, which attack the free bases, the nucleosides or the nucleotides (Fig.). Uric acid is then produced by the action of xanthine oxidase (EC 1.2.3.2), which is the key enzyme of aerobic P.d. In humans and apes, the uric acid is excreted largely unchanged. In most reptiles and mammals, it is oxidized to allantoin by uricase (EC 1.73.3) (uricolysis). 

---