D-Amino-acid oxidase

In another procedure, D-amino acid oxidase (52) is usehil to produce L-amino acids from DL-amino acids. a-Ketocarboxylic acids which are formed by the action of enzymes on D-amino acids, are aminated to form L-amino acids by coupling through the action of amino acid aminotransferases (53). 

L-methionine DL-methionine + Asp D-amino acid oxidase + transaminase Trigonopsis variabilis ... 

D-amino acid oxidase D-amino acids a-keto acids +NH3 2FAD U2 H2U2... 

Riboflavin-5 -Adenosine Diphosphate. Riboflavin-5 -adenosine diphosphate [146-14-5] (flavin—adenine dinucleotide, FAD), C27H33N9O15P2 (2), mol wt 785.56, was first isolated in 1938 from the D-amino acid oxidase as its prosthetic group (95), where it was postulated to be... 

D-Amino acid oxidase will oxidase only serine having the R configuration at C-2. Glycolate oxidase will remove only the prv-R hydrogen of glycolic acid Does the product (0=CHC02H) contain tritium Explain your reasoning. 

Riboflavin was first isolated from whey in 1879 by Blyth, and the structure was determined by Kuhn and coworkers in 1933. For the structure determination, this group isolated 30 mg of pure riboflavin from the whites of about 10,000 eggs. The discovery of the actions of riboflavin in biological systems arose from the work of Otto Warburg in Germany and Hugo Theorell in Sweden, both of whom identified yellow substances bound to a yeast enzyme involved in the oxidation of pyridine nucleotides. Theorell showed that riboflavin 5 -phosphate was the source of the yellow color in this old yellow enzyme. By 1938, Warburg had identified FAD, the second common form of riboflavin, as the coenzyme in D-amino acid oxidase, another yellow protein. Riboflavin deficiencies are not at all common. Humans require only about 2 mg per day, and the vitamin is prevalent in many foods. This vitamin... 

Figure 5.4 Deracemization of a-amino acids using D-amino acid oxidase in combination with sodium borohydride.

Henn, SW Ackers, GK, Molecular Sieve Studies of Interacting Protein Systems. V. Association of Subunits of D-Amino Acid Oxidase Apoenzyme, Biochemistry 8, 3829, 1969. 

Alonso J, JL Barredo, B Diez, E Mellado, E Salto, JL Garcia, E Cortes (1998) D-amino-acid oxidase gene from Rhodotorula gracilis (Rhodosporidium toruloides) ATCC 26217. Microbiology (UK) 144 1095-1101. 

Hamase, K., Inoue, T., Morikawa, A., Konno, R., Zaitsu, K. (2001). Determination of free d-proline and D-leucine in the brains of mutant mice lacking D-amino acid oxidase activity. Anal. Biochem. 298, 253-258. 

R. Fernandez-Lafuente, V. Rodriguez, and J.M. Guisan, The coimmobilization of D-amino acid oxidase and catalase enables the quantitative transformation of D-amino acids (D-phenylalanine) into alpha-keto acids (phenylpyruvic acid). Enz. Microb. Technol. 23, 28-33 (1998). 

Klein and Olsen126 studied the action of kojic acid on the enzymic oxidation of amino acids by the liver and kidney of rats. Low concentrations of kojic acid in vitro inhibited the oxidation of a number of D-amino acids, L-phenylalanine, and a few related compounds. Kojic acid was found to compete with D-amino acid oxidase for the substrate. 

Chumakov, I., Blumenfeld, M., Guerassimenko, O. et al. Genetic and physiological data implicating the new human gene G72 and the gene for D-amino acid oxidase in schizophrenia. Proc. Nat. Acad. Sci. USA 99 13675-13680, 2002. 

DAAO D-amino acid oxidase (iii) guanylyl cyclase... 

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.

D-Amino acid oxidase 1.4.3.3 D-Amino acids/oxygen Ammonia Oxygen uptake... 

The method described for the assay of D-amino acid oxidase in Procedure 8.4 illustrates the use of the Gilson respirometer in a gasometric assay. 

Enzymes are extremely useful analytical tools, primarily because of their specificity. Currently a very large number of enzymes are available commercially in various forms of purity and, although their cost is often high, only small amounts are usually required. Procedure 8.8 describes a fixed time method for the quantitation of D-alanine using D-amino acid oxidase. 

Glutamate dehydrogenase D-amino acid oxidase Standard solution D-alanine... 

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. 

Flavin Coenzymes.—5-Deazaflavin-adenine dinucleotide (2) can be prepared from the 5-deazaFMN,21 using a FAD pyrophosphorylase from rat liver.22 When the apoprotein of D-amino-acid oxidase from pig kidney is reconstituted with (2), no oxidation of D-alanine is observed, although the flavin chromophore in the reconstituted enzyme is reduced on the addition of DL-amino-acids.22 This has been interpreted as indicating that hydrogen transfer from the amino-acid to (2) can still... 

Many of the amino acids originally tested by Krebs were racemic mixtures. When naturally occurring L-amino acids became available the oxidase was found to be sterically restricted to the unnatural, D series. [D-serine occurs in worms free and as D-phosphoryl lombricine (Ennor, 1959)]. It could not therefore be the enzyme used in the liver to release NH3 in amino acid metabolism. D-amino acid oxidase was shown by Warburg and Christian (1938) to be a flavoprotein with FAD as its prosthetic group. A few years later Green found an L-amino acid oxidase in liver. It was however limited in its specificity for amino acid substrates and not very active—characteristics which again precluded its central role in deamination. 

An alternative two-step biocatalytic route, first developed at Glaxo in the 1970s, utilized a D-amino acid oxidase and an amidase to provide 7-ACA under physiological conditions (Scheme 1.12). This process has since been established in several companies, with minor modifications. In fact, 7-ACA was manufactured by GSK at Ulverston (Cumbria, UK) using both the chemical and biocatalytic processes in parallel for a period of 2 years during which time the environmental benefits of the biocatalytic process were assessed (see Section 1.6). 

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