Hydroxylases steroid, microsomal

Figure 11-6. Cytochrome P450 hydroxylase cycle in microsomes. The system shown is typical of steroid hydroxylases of the adrenal cortex. Liver microsomal cytochrome P450 hydroxylase does not require the iron-sulfur protein FejSj. Carbon monoxide (CO) inhibits the indicated step.

The 17- and 21-hydroxylase enzymes are associated with microsomes, whereas the ll- -hydroxylase has a mitochondrial origin. Since the last-named enzyme is not detectable in other steroid-producing tissues, the term 11-oxygenated steroids is considered synonymous with adrenal steroids. Aldosterone synthesis involves an essential 18-hydroxylation step catalyzed by P450d8 with corticosterone as the precursor this reaction also takes place within the mitochondria. 

Spironolactone exhibits antiandrogenic effects in males and females. It decreases testosterone biosynthesis by inhibiting steroid 17a-monooxygenase (17a-hydroxylase) activity, possibly secondary to destruction of microsomal cytochrome P-450 in tissues with high steroid 17a-monooxygenase activity (testes, adrenals) [65],... 

Waxman DJ, Attisano C, Guengerich FP, et al. Human liver microsomal steroid metabolism identification of the major microsomal steroid hormone 6P-hydroxylase cytochrome P-450 enzyme. Arch Biochem Biophys 1988 263 424-436. 

The same electron transfer system operates in the 22-, 20-, and 18-hydroxylations as well as in the 11 -hydroxylation. Further, the identical electron transfer system appears to be pertinent to the system of steroid hydroxylation in testis and ovary. 22-, 20-, 18-, and 11 p-hydroxy-lases are all distributed in the mitochondrial fraction of adrenal cortex, whereas 21- and 17-hydroxylases are located in the microsomal fraction. The electron transfer system in the microsomal steroid hydroxylation has not yet been established. 

The cytochome P-450 systems of the steroid producing tissues have many characteristics in common with those of the liver and other xenobiotic-metabolizing tissues. They require NADPH and molecular oxygen, and contain a flavoprotein, NADPH cytochrome P-450 reductase. Unlike the systems in the liver, however, the steroid hydroxylases are associated with both the endoplasmic reticulum and the mitochondria and are quite specific for the different reactions that they catalyze. Thus, adrenal microsomes... 

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. 

Metabolism studies. GC-MS is a powerful technique for following and identifying the metabolic products from the in vitro incubation of tissue preparations with steroid substrates. Examples of such studies include the 16a-hydroxylation of 18-hydroxydeoxycorticosterone by human adrenal gland [254], the eiromatization of 3jS,15, 16 -trihydroxy-5-androsten-17-one by placental homogenates [255], and the demonstration of 1/3, 12/3, 6a and 6/3 hydroxylase enzyme activities in microsomal preparations of human foetal hepatic tissue [256]. In the latter study, testosterone was used as substrate and in addition to the hydroxylated metabolites isolated, several other testosterone derivatives indicated the presence of 3a, 3/3 and 17/3-hydroxysteroid oxidoreductase in the adrenal gland preparation. 

Rat liver microsomes hydroxylate 5/8-cholestane-3a ,7a,12Q -triol at C-25 and C-26 both activities are dependent on cytochrome P450 and there is some evidence that different types of the latter are involved. A mitochondrial steroid 24-hydroxylase that accepts 3a,7a,12a-trihydroxy-5/3-cholestanoic acid has been extracted from rat liver apparently this is not a mixed-function oxidase although the presence of oxygen was obligatory for its action. Bile acids hydroxylated at C-23 have been formed from sodium cholate and deoxycholate in preparations from Viperinae species and a steroid-12ct-hydroxylase from liver microsomes has been studied.Sitosterol has been confirmed to be a precursor of C24 and C29 bile acids in mammalian liver, and here hydroxylation at C-26 precedes that at C-7. ° "... 

Improved methodology for the rapid assay of hepatic HMG-CoA reductase has been described and new and simplified assays are available for cholesterol 7a-hydroxylase, 4-methylsterol oxidase, 3/3-hydroxy-steroid dehydrogen-ase, the biosynthesis of bile acids, and microsomal cholesterol levels.The rate of biosynthesis of gibberellins has been monitored by a bioassay based on /3-amyrin production of Amaranthus seeds. 

Occasionally, the cytochrome P-450 system converts some chemieals to reactive species with carcinogenic potential (e.g., polycyclic hydrocarbons). The hepatic microsomal cytochrome P-450 system is inducible by many of its substrates. The cytoehrome P-450 of adrenal cortical mitochondria is involved in steroid hydroxylase reactions, and this system contains iron-sulfur (Fc2S2) proteins. 

During bile acid biosynthesis, modifications to the cyclopentanophen-anthrene (steroid) nucleus are thought to precede the oxidation and cleavage of the cholesterol side chain. The first and rate-controlling step in bile acid synthesis is the 7o-hydroxylation of cholesterol (I) to form 7a-hydroxy-choles-terol (II) (Fig. 3). This step is catalyzed by cholesterol 7a-monooxygenase (cholesterol 7a-hydroxylase) (EC 1.14.13.17), a microsomal enzyme (M37). Further metabolism of 7a-hydroxy-cholesterol involves oxidation of the 3p-hydroxyl group and isomerization of the double bond from C-5,6 to C-4,5,... 

Most studies on substrate specificity of cholesterol 7a-hydroxylase have been performed with intact microsomes. Results of such studies may be difficult to interpret since the enzyme system is embedded in a lipoprotein membrane, and may not be directly accessible to potential substrates [59]. Thus, differences in the rate of 7a-hydroxylation of various steroids could be due to differences in the rate at which the substrate reaches the active site of. the enzyme rather than to differences in the intrinsic ability of the enzyme to interact catalytically with the substrate [59], Further, occurrence of 7a-hydroxylation of a certain steroid may not reflect the substrate specificity of cholesterol 7a-hydroxylase activity since different species of cytochrome P-450 are present in the microsomes. 

Esters of cholesterol cannot be directly attacked by the enzyme [60,61]. Rat liver microsomes catalyse 7a-hydroxylation of cholestanol at a rate comparable to that of cholesterol [62]. Since 7a-hydroxylation of cholestanol is stimulated after treatment with cholestyramine, it appears likely that the same enzyme is involved as in hydroxylation of cholesterol. Shght changes in the side chain lead to marked loss of activity [63,64]. Loss of a terminal methyl group reduces the rate to about 50% and addition of an ethyl group at C-24 (sitosterol) leads to almost complete loss of the activity. From a study with a great number of structurally closely related steroids, Aringer concluded that cholesterol 7a-hydroxylase requires a rather flat steroid (A", A or 5a) and an equatorial or quasi-equatorial hydroxyl group at C-3 [65]. 

The microsomal 26-hydroxylase in rat liver has a higher substrate specificity than the mitochondrial. Of a number of C27-steroids, only 5j8-cholestane-3a,7a,12a-triol, 5)8-cholestane-3a,7a-diol, 7a-hydroxy-4-cholesten-3-one and 7a,12a-dihydroxy-4-cholesten-3-one were 26-hydroxylated to a significant extent [126]. In addition to hydroxylation in the 26 position, 5)8-cholestane-3a,7a,12a-triol was hydroxylated by the microsomal fraction of rat liver in the 23, 24 , 24/8 and 25 positions [40]. The hydroxylation in the 25 position was about as efficient as that in the 26 position. 

Conclusive evidence that a species of cytochrome P-450 was involved in the hydroxylation was presented by Okuda et al., who showed that the photochemical action spectrum for reversal of the carbon monoxide inhibition of 26-hydroxylation of 5)8-cholestane-3a,7a,12a-triol in rat liver exhibited a maximum at 450 nm [134]. Pedersen et al. [135] and Sato et al. [136] reported simultaneously that small amounts of cytochrome P-450 could be solubilized from the inner membranes of rat liver mitochondria that was active towards cholesterol as well as 5)8-cholestane-3a,7a,12a-triol in the presence of ferredoxin, ferredoxin reductase and NADPH. The mechanism of hydroxylation is thus the same as that operative in the biosynthesis of steroid hormones in the adrenals and in the la-hydroxylation of 25-hydroxyvitamin D in the kidney (Fig. 8). The liver mitochondrial cytochrome P-450 was not active in the presence of microsomal NADPH-cytochrome P-450 reductase [135,136]. Ferredoxin reductase as well as ferredoxin were active regardless of whether they were isolated from rat liver mitochondria or bovine adrenal mitochondria [133]. The partially purified cytochrome P-450 had a carbon monoxide difference spectrum similar to that of microsomal cytochrome P-450 from liver microsomes and adrenal mitochondria. In the work by Pedersen et al. [133], the concentration of mitochondrial cytochrome P-450 in rat liver mitochondria from untreated rats was calculated to be only about 0.1 nmole/mg protein. Treatment of rats with phenobarbital increased the specific content of cytochrome P-450 in the mitochondria more than 2-fold, without significant increase in the 26-hydroxylase activity. The carbon monoxide spectrum of the reduced cytochrome P-450 solubilized from liver mitochondria of phenobarbital-treated rats exhibited a spectral shift of about 2 nm as compared to the corresponding spectrum obtained in analysis of preparations from untreated rats. This was taken as evidence that more than one species of cytochrome P-450 was present in the preparation. It was later shown by Pedersen et al. [137] and Bjbrkhem et al. [138] that the preparation was also able to catalyse 25-hydroxylation of vitamin D3 and that different enzymes are involved in... 

Morgan, E.T., C. MacGeoch, and J.-A. Gustafsson (1985). Hormonal and developmental regulation of expression of the hepatic microsomal steroid 16alpha-hydroxylase cytochrome P-450 apoprotein in the rat. J. Biol. Chem. 260, 11895-11898. 

While these cyclic spectral changes in conjunction with the hydrox-ylation of the substrate can be most clearly demonstrated in the steroid 21-hydroxylase system of adrenocortical microsomes, substrate-produced spectral changes have been described in several mixed-function oxidase systems, such as the induced drug hydroxylations of rat liver microsomes (27) and the 11 -hydroxylation of cortexone by intact mitochondria (2, 12) or by the supernatant Si of mitochondrial sonicates (5). 

The apparent substrate dissociation constant Kg for cortexone of 4 X 10 M is quite similar to that of 17-hydroxyprogesterone in steroid 21-hydroxylase preparations from bovine adrenocortical microsomes (7). This distinguishes the adrenal steroid hydroxylases from the drug hydroxylases of rat liver where values of about lO M were observed (27). 

Nakajin S, Hall PF. Microsomal cytochrome P-450 from neonatal pig testis. Purification and properties of a C-21 steroid side-ohain cleavage system (17a-hydroxylase-C17,20-lyase). J Biol Chem 1981 256 3871-3876. 

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