Flavoprotein enzymes

Flavoprotein enzymes contain flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) as prosthetic groups. FMN and FAD are formed in the body from the vitamin riboflavin (Chapter 45). FMN and FAD are usually tighdy—but not covalendy—bound to their respecdve apoenzyme proteins. Metalloflavopro-teins contain one or more metals as essential cofactors. 

Examples of flavoprotein enzymes include L-amino acid oxidase, an FMN-finked enzyme found in kidney with general specificity for the oxidadve deamination of... 

MAO is a flavoprotein enzyme that is found on the outer membrane of mitochondria. It oxidatively deami nates short-chain monoamines only, and it is not part of the DM MS. ATP is involved in the transfer of reducing equivalents through the mitochondrial respiratory chain, not the microsomal system. 

Molybdenum (Mo) is present in all plant, human, and animal tissues, and is considered an essential micronutrient for most life forms (Schroeder et al. 1970 Underwood 1971 Chappell and Peterson 1976 Chappell et al. 1979 Goyer 1986). The first indication of an essential role for molybdenum in animal nutrition came in 1953 when it was discovered that a flavoprotein enzyme, xanthine oxidase, was dependent on molybdenum for its activity (Underwood 1971). It was later determined that molybdenum is essential in the diet of lambs, chicks, and turkey poults (Underwood 1971). Molybdenum compounds are now routinely added to soils, plants, and waters to achieve various enrichment or balance effects (Friberg et al. 1975 Friberg and Lener 1986). 

Nicotinic acid derivatives occur in biologic materials as the free acid, as nicotinamide, and in two coenzymatic forms nicotinamide adenine dinucleotide (NAD), and nicotinamide adenine dinucleotide phosphate (NADP). These coenzymes act in series with flavoprotein enzymes and, like them, are hydrogen acceptors or, when reduced, donors. Several plants and bacteria use a metabolic pathway for the formation of nicotinic acid that is different from the tryptophan pathway used by animals and man (B39). 

Riboflavin (vitamin B2 6.18) consists of an isoalloxazine ring linked to an alcohol derived from ribose. The ribose side chain of riboflavin can be modified by the formation of a phosphoester (forming flavin mononucleotide, FMN, 6.19). FMN can be joined to adenine monophosphate to form flavin adenine dinucleotide (FAD, 6.20). FMN and FAD act as co-enzymes by accepting or donating two hydrogen atoms and thus are involved in redox reactions. Flavoprotein enzymes are involved in many metabolic pathways. Riboflavin is a yellow-green fluorescent compound and, in addition to its role as a vitamin, it is responsible for the colour of milk serum (Chapter 11). 

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. 

The functions of flavoprotein enzymes are numerous and diversified.151-1533 A few of them are shown in Table 15-2 and are classified there as follows (A) oxidation of hemiacetals to lactones, (B) oxidation of alcohols to aldehydes or ketones, (C) oxidation of amines to imines, (D) oxidation of carbonyl compounds or carboxylic acids to a,(3-unsaturated compounds,... 

The pKa assignments, which have been controversial, are discussed in Chapter 7. This disulfide loop is reduced by NADPH through the action of the flavoprotein enzyme thioredoxin reductase. 

In the catalytic cycle of CYP, reducing equivalents are transferred from NADPH to CYP by a flavoprotein enzyme known as NADPH-cytochrome P450 reductase. The evidence that this enzyme is involved in CYP monooxygenations was originally derived from the observation that cytochrome c, which can function as an artificial electron acceptor for the enzyme, is an inhibitor of such oxidations. This reductase is an essential component in CYP-catalyzed enzyme systems reconstituted from purified components. Moreover antibodies prepared from purified reductase are inhibitors of microsomal... 

Walsh C, Cromartie T, Marcotte P, et al. Suicide substrates for flavoprotein enzymes. Meth Enzymol 1978 53 437 48. 

In addition to liver aldehyde dehydrogenase, a number of other enzymes present in the soluble fraction of liver homogenates will oxidize aldehydes and certain N-heterocyclic compounds. Among these are aldehyde oxidase and xanthine oxidase (see below), both flavoprotein enzymes containing molybdenum. These enzymes catalyze the oxidation of aldehydes formed by the deamination of endogenous amines by amine oxidases. 

Indoleacetic acid is degraded in plants by a specific indoleacetic acid oxidase. This is a light-activatable flavoprotein enzyme coupled through hydrogen peroxide to a peroxidase (285 but cf. 463a, 805b). It apparently uses phenols as cofactors (296) but can be inhibited by polyphenols (305). The product of the reaction is still unidentified (836). 

A limited quantity of D-lactate is converted to pyruvate by a mitochondrial flavoprotein enzyme D-2-hydroxy acid dehydrogenase. Thus, the development of D-lactate acidosis requires excessive production of D-lactate and an impairment in its metabolism. The clinical manifestations of D-lactic acidosis are characterized by episodes of encephalopathy after ingestion of foods containing carbohydrates. 

Regeneration of the ribonucleotide reductase is accomplished in Escherichia coli and in mammals by thioredoxin, a dithiol polypeptide (M.W. 12,000) coenzyme, which also plays a role in other protein disulfide reductase reactions. In thioredoxin, two cysteine residues in the sequence -Cys-Gly-Pro-Cys are converted to cystine. Reduced thioredoxin is regenerated by thioredoxin reductase, a flavoprotein enzyme that uses NADPH + H+. 

Those include C-, N- and 5-oxidations, N-, 0- and 5-deaIkylation, deaminations, and certain dehalogenations. Under anaerobic conditions, it can also catalyze reductive reactions. The CYP monooxygenase system is a multien-zymatic complex constituted by the CYP hemoprotein, the flavoprotein enzyme NADPH CYP reductase, and the unsatnrated phospholipid phosphatidylcholine. The isoforms involved in xenobiotic metabolism are membrane bonnd enzymes situated in the endoplasmic reticnlnm. After... 

Our own studies, in which we have investigated H-tunneling in a number of quinoprotein and flavoprotein enzymes, have provided evidence consistent with H-transfer by quantum tunneling from the vibrational ground state of the reactive C-H bond of the substrate, and either H-tunneling in which the KIE is temperature independent - we interpret this to correspond to the absence of gated motion (that is no compression of the transfer distance by substrate and/or protein flue-... 

We have recently reviewed our work on quinoprotein enzymes elsewhere [42]. In this chapter we review our studies of H-tunneling in flavoprotein enzymes. 

Table 13.1. Tunneling regimes and associated parameters in various flavoprotein enzymes.

The first three reactions are catalyzed by a trifunctional protein which contains carbamoyl-phosphate synthetase II, aspartate carbamoyltransferase and dihydro-orotase. This set of reactions begins with the synthesis of carbamoyl phosphate followed by its condensation with aspartic acid. The third step involves the closure of the ring through the removal of water by the action of dihydro-orotase to yield dihydro-orotate. The fourth enzyme, dihydro-orotate oxidase, oxidizes dihydro-orotate to orotate and is a mitochondrial flavoprotein enzyme located on the outer surface of the inner membrane and utilizes NAD" " as the electron acceptor. The synthesis of UMP from orotate is catalyzed by a bifunctional protein which comprises orotate PRTase and orotidine 5 -phosphate (OMP) decarboxylase. The former phosphoribosylates orotate to give OMP the latter decarboxylates OMP to UMP, the immediate precursor for the other pyrimidine nucleotides. It is interesting to note that whereas five molecules of ATP (including the ATP used in the synthesis of PRPP) are used in the de novo synthesis of IMP, no net ATP is used in the de novo synthesis of UMP. In de novo pyrimidine synthesis, two ATP molecules are used to synthesize carbamoyl phosphate and one ATP is needed to synthesize the PRPP used by orotate PRTase but 3 ATPs... 

Xanthine oxidase is another flavoprotein which can catalyse nitroreduction in hypoxia with redox-controlled rates [149,150]. Figure 5 shows that the redox dependencies of nitroreduction by free flavin or by three different flavoprotein enzymes are not very different, and of the same order as that expected for electron-transfer reactions [100,151]. Cytotoxicity has a similar redox dependence, in both oxic [96] and hypoxic [97] conditions. 

---