Cofactor requirements, microsomal enzymes

Indicine IV-oxide (169) (Scheme 36) is a clinically important pyrrolizidine alkaloid being used in the treatment of neoplasms. The compound is an attractive drug candidate because it does not have the acute toxicity observed in other pyrrolizidine alkaloids. Indicine IV-oxide apparently demonstrates increased biological activity and toxicity after reduction to the tertiary amine. Duffel and Gillespie (90) demonstrated that horseradish peroxidase catalyzes the reduction of indicine IV-oxide to indicine in an anaerobic reaction requiring a reduced pyridine nucleotide (either NADH or NADPH) and a flavin coenzyme (FMN or FAD). Rat liver microsomes and the 100,000 x g supernatant fraction also catalyze the reduction of the IV-oxide, and cofactor requirements and inhibition characteristics with these enzyme systems are similar to those exhibited by horseradish peroxidase. Sodium azide inhibited the TV-oxide reduction reaction, while aminotriazole did not. With rat liver microsomes, IV-octylamine decreased... 

Flavanone 3 -hydroxylase (F3 H ECl.14.13.21 CYP75B) activity was initially identified in microsomal preparations of golden weed (Haplopappus gracilis) [110]. E3 H from irradiated parsley cell cultures was later biochemically analyzed and characterized as a cytochrome P450 having an absolute requirement for NADPH and molecular oxygen as cofactors [111]. The enzyme has been shown to have activity with flavanones, flavones, dihydroflavonols, and flavonols, but does not appear to have activity with anthocyanidins [111]. The first cDNA clone for E3 H was isolated from Petunia [112]. It has been suggested that E3 H may serve as an anchor for the proposed flavonoid multi-enzyme complex on the cytosolic surface of the endoplasmic reticulum [44]. 

Fig. 13. Opening of the 19-cyclopropane ring during sterol biosynthesis in photosynthetic organisms. This is catalysed by a microsomal enzyme that appears to require no cofactors and generates a sterol product that possesses a lOp-methyl group and a A -double bond.

The C. alpina microsomal A12 acetylenase and A12 desaturase activities were characterized with resepect to cofactor requirement and inhibitors (Table 1). Both enzymes showed similar cofactor requirements and responded similarly to the different inhibitors indicating that they may both belong to the same type of monoxygenases. It is pertinent to note that the acetylenase reaction is a desaturation. 

Microsomes are widely used to study the metabolism of xenobiotics. Enzymes can be chararacterized on the basis of their requirement for cofactors (e.g., NADPH, UDPGA), and their response to inhibitors. Kinetic studies can be carried out, and kinetic constants determined. They are very useful in studies of comparative metabolism, where many species not available for in vivo experiment can be compared with widely investigated laboratory species such as rats, mice, feral pigeon, Japanese quail, and rainbow trout. 

Microsomal flavin-containing monooxygenases. As well as the cytochromes P-450 MFO system, there is also a system, which uses FAD. This flavin-containing monooxygenase or FMO enzyme system is found particularly in the microsomal fraction of the liver, and the monomer has a molecular weight of around 65,000. Each monomer has one molecule of FAD associated with it. The enzyme may accept electrons from either NADPH or NADH although the former is the preferred cofactor. It also requires molecular oxygen, and the overall reaction is as written for cytochromes P-450 ... 

Depending on the in vitro technique employed, only certain types of metabolic reactions are possible or require the addition of specific cofactors for reactions to occur. For example, microsomes do not have the ability to form sulfate metabolites due to the lack of both the sulfotransferase enzyme and the necessary cofactor. Table 3 illustrates the metabolic events that are possible within each in vitro system, assuming the necessary cofactors are added in the appropriate concentrations. The location of these enzymes will be important to remember when choosing an in vitro technique to use and which products (metabolites) to expect from each system. 

The use of microsomes along with UDPGA as a cofactor assay to measure UGT enzyme activity has been hampered historically by the fact that this enzymatic activity in microsomes is often in a latent form and requires activation by physical or detergent-induced disruption of the membrane matrices. Recently, a generic method involving the addition of the pore-forming peptide alamethicin to overcome the latency exhibited by this enzyme system has been described.99 The inclusion of alamethicin seems to provide a more consistent method of assessing UGT enzyme activity. 

Others such as pig liver microsomal FAD-containing monooxygenase have to be isolated from tissue with very low yields141 or like hydrocarbon monooxygenase from Pseudomonas oleovorans 12 131 require several protein components and cofactors, substantially limiting the use of these enzymes for the production of oxidized sulfur compounds. 

Oxidative cleavage of the O-alkyl linkage in glycerolipids is catalyzed by a microsomal tetrahydropteridine (Pte-H4)-dependent alkyl monooxygenase (Fig. 12) (T.-C. Lee, 1981). The required cofactor, Pte H4, is regenerated from Pte-Hj by an NADPH-linked pteridine reductase, a cytosolic enzyme. Oxidative attack on the ether bond in lipids is similar to the enzymatic mechanism described for the hydroxylation of phenylalanine. Fatty aldehydes produced via the cleavage reaction can be either oxidized to the corresponding acid or reduced to the alcohol by appropriate enzymes. 

An enzyme from the flowers of Sinningia cardinalis Reichsteinia cardinalis, Gesneriaceae) has hydrolase activity associated with microsomal fractions and requires NADPH as an essential cofactor (hydrolyase activity II). This enzyme converts naringenin (10) and apigenin (5) to eriodictyol (17) and luteolin (4), respectively (Dewick, 1989). The flavone synthase activity of this enzyme was abolished completely by treatment with the cytochrome P-450 inhibitor ancymidol, but the flavonoid 3 -monooxygen-ase activity was not altered. 

Three indoleacetaldehyde reductases were purified from cucumber seedlings [ 1,4]. The enzyme requiring NADH as a cofactor occurred in the cytosol one of the two NADPH-specific reductases was associated with a microsomal fraction. The latter reduced phenylacetaldehyde at about half the rate observed for indoleacetaldehyde and exhibited minor activity on some of the aliphatic aldehydes tested. The NADH-requiring enzyme acted only on indoleacetaldehyde and phenylacetaldehyde. None of the three enzymes would catalyze the reverse oxidation of tryptophol. 

Elongating systems in mitochondria. Brain mitochondria are able to elongate acyl-CoA in the presence of acetyl-CoA (16). Pal-mityl-CoA is elongated by acetyl-CoA in the presence of NADPH and NADH providing mainly stearic acid. Similar patterns for enzymatic activity, cofactors, optimal pH and substrate requirements are found for stearyl-CoA elongation presuming that the same enzyme systems elongate both acyl-CoA (contrary to microsomes) (17). 

Secondly, one wants a structure which is a good acceptor for an acyl group but a poor one for a phosphoryl group. This could be an SH, a reactive OH, or the NH of an imidazole or amide group. Among the known vitamins and cofactors there are, of course, several with the necessary structural requirements outlined above. The first to come to mind is CoA, but so far it has not been possible to demonstrate a CoA requirement for amino acid incorporation into mammalian or plant microsomes. Nevertheless this vitamin seems to be essential for the incorporation of amino acids into the proteins of hen oviducts (56) and it does, of course, the job of displacing activated fatty acids from their activating enzymes (170). Vitamin Bi2 also fits the structural requirements and it has, indeed, been claimed to be essential for amino acid activation and subsequent incorporation into rat liver microsomes (i07, 178, 179), but this requirement has not yet been confirmed by other authors (180,181). 

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