Flavoprotein oxidase substrate specificity

Amine oxidation. As well as the microsomal enzymes involved in the oxidation of amines, there are a number of other amine oxidase enzymes, which have a different subcellular distribution. The most important are the monoamine oxidases and the diamine oxidases. The monoamine oxidases are located in the mitochondria within the cell and are found in the liver and also other organs such as the heart and central nervous system and in vascular tissue. They are a group of flavoprotein enzymes with overlapping substrate specificities. Although primarily of importance in the metabolism of endogenous compounds such as 5-hydroxy try pt-amine, they may be involved in the metabolism of foreign compounds. 

Flavins are very versatile redox coenzymes. Flavopro-teins are dehydrogenases, oxidases, and oxygenases that catalyze a variety of reactions on an equal variety of substrate types. Since these classes of enzymes do not consist exclusively of flavoproteins, it is difficult to define catalytic specificity for flavins. Biological electron acceptors and donors in flavin-mediated reactions can be two-electron acceptors, such as NAD+ or NADP+, or a variety of one-electron acceptor systems, such as cytochromes (Fe2+/ Fe3+) and quinones, and molecular oxygen is an electron acceptor for flavoprotein oxidases as well as the source of oxygen for oxygenases. The only obviously common aspect of flavin-dependent reactions is that all are redox reactions. 

DAAO is one of the most extensively studied flavoprotein oxidases. The homodimeric enzyme catalyzes the strictly stere-ospecihc oxidative deamination of neutral and hydrophobic D-amino acids to give a-keto acids and ammonia (Fig. 3a). In the reductive half-reaction the D-amino acid substrate is converted to the imino acid product via hydride transfer (21). During the oxidative half-reaction, the imino acid is released and hydrolyzed. Mammalian and yeast DAAO share the same catalytic mechanism, but they differ in kinetic mechanism, catalytic efficiency, substrate specificity, and protein stability. The dimeric structures of the mammalian enzymes show a head-to-head mode of monomer-monomer interaction, which is different from the head-to-tail mode of dimerization observed in Rhodotorula gracilis DAAO (20). Benzoate is a potent competitive inhibitor of mammalian DAAO. Binding of this ligand strengthens the apoenzyme-flavin interaction and increases the conformational stability of the porcine enzyme. 

Fatty Acyl CoA Oxidation. The two flavoproteins which have been isolated that oxidize fatty acyl CoA compounds differ in cofactors as well as in substrate specificity. The yellow enzsrme, acting on longer fatty acid derivatives, may contain iron in addition to FAD. The green enzyme, acting on 3- to 8-carbon chains, contains 2 atoms of copper per FAD. As in the case of other metal-flavins, the copper appears to be required only for transfer to cytochrome c. The implicit role ascribed to the metal is difficult to reconcile with the recent report of additional ffavoproteins with low metal content required to mediate between the fatty acyl CoA oxidases and either dyes or cytochrome c. These enzymes, called electron-transferring ffavoprotein or ETF, exhibit peaks near 375 and 437 m/i with a shoulder at 460 m/i. 

The highest L-amino acid oxidase activity is found in the venoms of a variety of snakes, the enzyme being present to the extent of up to 3% of dried, whole venom in some species. Crystalline enzymes (both flavoproteins) have been isolated from the venoms of water moccasin Ancistrodonpiscivorus) and rattle snake Crotalus adamanteus) and shown to be similar with respect to molecular weight (130,000) and flavin content (2 moles FAD/mole protein). The enzymes are not substrate-specific but are more active with the L-isomers of methionine, leucine, tryptophan, phenylalanine and tyrosine than with other L-amino acids. The reactions catalysed by L-amino acid oxidase are formally the same as those discussed in section V.A (reactions 23-26) but, as is the case for the D-amino acid oxidase, the mechanism of... 

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. 

Oi-, OH or OH+), a similar mode of action can be taken into consideration for the native flavoproteins. The specific action of different flavin oxidases, dehydrogenases and hydroxylases can most likely be attributed to specific proteins which are bound with the flavin moiety and are in a position to dictate the particular breakdown of HFIOOH. For example, the oxidases were thought to react specifically, yielding oxidized flavin and H2O2, while the dehydrogenases were supposed to yield both flavin and superoxide radicals (HF1- + HO 2)- The hydroxylases were assumed to account for the reaction of one of the activated oxygen intermediates with the respective substrate. 

In contrast, CaM s activation of NO synthesis and substrate-independent NADPH oxidase activity did appear to involve flavin-to-heme electron transfer, because these reactions were not activated in apo-NOS and were blocked in native NOS by agents that prevent heme iron reduction (Abu-Soud et al., 1994a). We conclude that CaM activates neuronal NOS at two points (Fig. 2) electron transfer into the flavins and interdomain electron transfer between the flavins and heme. Activation at each point is associated with an up-regulation of domain-specific catalytic functions. The dual regulation by CaM is unique and represents a new means by which electron transfer can be controlled in a metal-containing flavoprotein. 

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.). 

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