Hydroxylases, mixed function oxidases

FIGURE 25.42 The mixed-function oxidase activity of 7ff-hydroxylase. 

Cytochrome P450 2C19, also termed S-mephenytoin hydroxylase, is a mixed-function oxidase localized in the endoplasmic reticulum which is responsible for the biotransformation of S-mephenytoin, some barbiturates, almost all proton pump inhibitors such as omeprazole, diazepam and others. 

Monooxygenases (Mixed-Function Oxidases, Hydroxylases) Incorporate Only One Atom of Molecular Oxygen Into the Substrate... 

The oxidation of carotenes results in the formation of a diverse array of xanthophylls (Fig. 13.7). Zeaxanthin is synthesised from P-carotene by the hydroxylation of C-3 and C-3 of the P-rings via the mono-hydroxylated intermediate P-cryptoxanthin, a process requiring molecular oxygen in a mixed-function oxidase reaction. The gene encoding P-carotene hydroxylase (crtZ) has been cloned from a number of non-photosynthetic prokaryotes (reviewed by Armstrong, 1994) and from Arabidopsis (Sun et al, 1996). Zeaxanthin is converted to violaxanthin by zeaxanthin epoxidase which epoxidises both P-rings of zeaxanthin at the 5,6 positions (Fig. 13.7). The... 

Tyrosine hydroxylase is the rate-limiting enzyme for the biosynthesis of catecholamines. Tyrosine hydroxylase (TH) is found in all cells that synthesize catecholamines and is a mixed-function oxidase that uses molecular oxygen and tyrosine as its substrates and biopterin as its cofactor [1], TH is a homotetramer, each subunit of which has a molecular weight of approximately 60,000. It catalyzes the addition of a hydroxyl group to the meta position of tyrosine, thus forming 3,4-dihydroxy-L-phenylalanine (l-DOPA). 

The CYP enzymes active in phase I reactions are often oxidases or hydroxylases, sometimes called mixed function oxidase (MFO). An oxidase enzyme introduces into the substrate (i.e. the unwanted compound) both atoms of an oxygen molecule whilst... 

BENZO(a)PYRENE HYDROXYLASE (AHH) ACTIVITY OF A RECONSTITUTED MIXED-FUNCTION OXIDASE SYSTEM CONTAINING HEPATIC CYTOCHROME P-448 FROM DBA-TREATED MALE SKATES... 

The marine environment acts as a sink for a large proportion of polyaromatic hydrocarbons (PAH) and these compounds have become a major area of interest in aquatic toxicology. Mixed function oxidases (MFO) are a class of microsomal enzymes involved in oxidative transformation, the primary biochemical process in hydrocarbon detoxification as well as mutagen-carcinogen activation (1,2). The reactions carried out by these enzymes are mediated by multiple forms of cytochrome P-450 which controls the substrate specificity of the system (3). One class of MFO, the aromatic hydrocarbon hydroxylases (AHH), has received considerable attention in relation to their role in hydrocarbon hydroxylation. AHH are found in various species of fish (4) and although limited data is available it appears that these enzymes may be present in a variety of aquatic animals (5,6,7,8). 

An effect of ozone on lung microsomes has been suggested by morpholine studies that indicated alterations in the endoplasmic reticulum, Biochemical evidence of an effect on microsomal enzymes was originally obtained in the studies of Palmer et who demonstrated that ozone exposure (0.75-10 ppm for 3 h) resulted in a decrease in activity of Syrian hamster lung benzopyrene hydroxylase, a mixed-function oxidase that depends on cytochrome P-450. No changes in hepatic activities of this enzyme were observed, and the results were similar in animals in which high activities of benzopyrene hydroxylase had been induced. The maximal effect was not observed until a few days after the single ozone exposure. Palmer et also reported a decrease in rabbit tracheobronchial mucosal benzopyrene hydroxylase activity after exposure to similar ozone concentrations. 

Putidaredoxin. Cushman et al. (36) isolated a low molecular iron-sulfur protein from camphor-grown Pseudomonas putida. This protein, putidaredoxin, is similar to the plant type ferredoxins with two irons attached to two acid-labile sulfur atoms (37). It has a molecular weight of 12,000 and shows absorption maxima at 327, 425 and 455 nm. Putidaredoxin functions as an electron transfer component of a methylene hydroxylase system involved in camphor hydroxylation by P. putida. This enzyme system consists of putidaredoxin, flavoprotein and cytochrome P.cQ (38). The electron transport from flavoprotein to cytochrome P.cq is Smilar to that of the mammalian mixed-function oxidase, but requires NADH as a primary electron donor as shown in Fig. 4. In this bacterial mixed-function oxidase system, reduced putidaredoxin donates an electron to substrate-bound cytochrome P. g, and the reduced cytochrome P. g binds to molecular oxygen. One oxygen atom is then used for substrate oxidation, and the other one is reduced to water (39, 40). 

Megaredoxin. Another example of a bacterial mixed-function oxidase was found in the steroid 15 6-hydroxylase system of Bacillus megaterium (41). This enzyme system consists of three proteins FMN-containing flavoprotein (megaredoxin reductase), iron-sulfur protein... 

Phenylalanine hydroxylase (also called phenylala-nine-4-monooxygenase) is one of a general class of enzymes called mixed-function oxidases (see Box 21-1), all of which catalyze simultaneous hydroxylation of a substrate by an oxygen atom of 02 and reduction of the other oxygen atom to H20. Phenylalanine hydroxylase... 

The conversion of cholesterol to bile acids is quantitatively the most important mechanism for degradation of cholesterol. In a normal human adult approximately 0.5 g of cholesterol is converted to bile acids each day. The regulation of this process operates at the initial biosynthetic step catalyzed by an enzyme in the endoplasmic reticulum, la-hydroxylase (fig. 20.18). The 7a-hydroxylase is one of a group of enzymes called mixed-function oxidases, which are involved in the hydroxylation of the sterol molecule at numerous specific sites. A mixed-function oxidase is an enzyme complex that catalyzes hydroxylation of a substrate with a concomitant production of H20 from a single molecule of 02- The 7a-hydroxylase is one of several enzymes referred to as cytochrome P450. 

Aryl hydrocarbon hydroxylase (AHH) is a complex mixed function oxidase enzyme which converts polycyclic aromatic hydrocarbons such as DMBA to more hydrophilic and readily excretable products. During this process metabolites that are more carcinogenic than the parent compound can be produced. The distribution of MFO enzymes, their activity, and the balance between conversion of procarcinogens to active carcinogens and their detoxification is probably a... 

Figure 5. Dietary protein and hepatic mixed function oxidase activity (cytochrome c-reductase, cytochrome P-450, aryl hydrocarbon hydroxylase). (Reproduced with permission from Ref. 71. Copyright 1983, American Dairy Science Association.)...

A great deal of information is now available about the properties, constitution and clinical manifestations of deficiencies of the hydroxylases involved in corticosteroid synthesis [42,52,53], and only a fraction of that information can be mentioned here. All the hydroxylases are mixed-function oxidases, requiring NADPH and 02, and some seem to be associated with cyts P-450, viz. cyt P-450 u/3, cyt P-4502i, cyt P-45018. The following equation represents the reaction catalysed ... 

Vitamin D that is taken up by the fiver is converted to 25-hydroxyvitamin D by a microsomal hydroxylase (Fig. 30-3). 25-Hydroxyvitamin D is the main circulating form of vitamin D in the serum and the best indicator of vitamin D status. Normal serum levels are 14-60 ng/mL (35-150 nmol/L). When serum calcium concentrations decline, 25-hydroxyvitamin D is converted to 1,25-dihydroxyvitmin D by la-hydroxylase, a mixed-function oxidase that is located in the inner mitochondrial membrane in kidney tissue and whose expression is regulated by parathyroid hormone (PTH). The main function of 1,25-dihydroxyvitamin D is to increase the intestinal absorption of dietary calcium and phosphorus. When serum concentrations of calcium and phosphorus are normal or when large doses of vitamin D are administered, 25-hydroxyvitamin D is metabolized to 24,25-dihydroxyvitamin D in the renal... 

The first enzyme activity (dihydrobiopterin reductase) catalyzes the transfer of hydrogen to dihydrobiopterin, which is thus reduced to tetrahydrobiopterin. The second enzyme activity is a hydroxylase containing two Fe3+ atoms, and this catalyzes the reduction of Oz such that one oxygen atom is incorporated into phenylalanine to form tyrosine and the second into water. At the same time tetrahydrobiopterin is oxidized to dihydrobiopterin. Phenylalanine hydroxylase is an example of a mixed-function oxidase. An inherited deficiency of phenylalanine hydroxylase results in the accumulation of phenylalanine that is not converted to tyrosine but is excreted as phenylpyruvate. This condition, which affects young infants, is known as phenylketonuria and is associated with severe mental retardation. 

As with the mixed-function oxidases involved in xenobiotic metabolism, the substrate specificity of the steroid hydroxylases is dictated, in part, by the existence of multiple forms of both microsomal and mitochondrial cytochrome P-450s and further opportunities for specificity are provided by the distinct localization of the various enzymes in either the mitochondria or the endoplasmic reticulum. 

Aryl hydrocarbon hydroxylase (AHH) is part of the microsomal mixed-function oxidase system involved in the detoxification of polycyclic aromatic hydrocarbons. In the HPLC assay developed for the AHH activity, benzo[a]pyrene (BaP) is used as the substrate, and the activity is determined by measuring the unreacted BaP during the reaction. 

Kynurenine Hydroxylase Kynurenine hydroxylase is an FAD-dependent mixed-function oxidase of the outer mitochondrial membrane, which uses NADPH as the reductant. The activity of kynurenine hydroxylase in the liver of riboflavin-deficient rats is only 30% to 50% of that in control animals, and deficient rats excrete abnormally large amounts of kynurenic and anthranilic acids after the administration of a loading dose of tryptophan, and, correspondingly lower amounts of quinolinate and niacin metabolites. Riboflavin deficiency may thus be a contributory factor in the etiology of pellagra when intakes of tryptophan and niacin are marginal (Section 8.5.1). 

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