Byh

FIGURE 2-28 Proposed scheme for the cyclic reduction and oxidation of cytochrome P450 in biological hydroxylations. The process of binding of the substrates, a steroid, and molecular 02 by the cytochrome P450 enzyme and transfer of the electrons is complex. The key participant is the hexacoordinate iron ion, which is bound to the planar tetrapyrrolic heme prosthetic group. Four of the iron valencies are taken up by binding with the four nitrogen atoms of the tetrapyrrole; the remaining two valencies are utilized to coordinate with other ligands (cysteinate and molecular oxygen) at right angles to the plane of the pyrrole ring structure. The strength or weakness of the bound ligands with respect to the nature of the uncharged or charged moiety bonding to the metal ion can greatly alter the physical properties of the hemoprotein. This alteration has been termed "low spin" or "high spin." Modified from Boyd, G. S. (1973). In "Biological Hydroxylation Mechanisms" (G. S. Boyd and R. M. S. Smellie, eds.), p. 1. Academic Press, New York. Copyright © 1973 The Biochemical Society, London.

FIGURE 2-28 Proposed scheme for the cyclic reduction and oxidation of cytochrome P450 in biological hydroxylations. The process of binding of the substrates, a steroid, and molecular 02 by the cytochrome P450 enzyme and transfer of the electrons is complex. The key participant is the hexacoordinate iron ion, which is bound to the planar tetrapyrrolic heme prosthetic group. Four of the iron valencies are taken up by binding with the four nitrogen atoms of the tetrapyrrole; the remaining two valencies are utilized to coordinate with other ligands (cysteinate and molecular oxygen) at right angles to the plane of the pyrrole ring structure. The strength or weakness of the bound ligands with respect to the nature of the uncharged or charged moiety bonding to the metal ion can greatly alter the physical properties of the hemoprotein. This alteration has been termed "low spin" or "high spin." Modified from Boyd, G. S. (1973). In "Biological Hydroxylation Mechanisms" (G. S. Boyd and R. M. S. Smellie, eds.), p. 1. Academic Press, New York. Copyright © 1973 The Biochemical Society, London.

terminal extensions. The N-terminal extension apparently serves as a leader sequence to facilitate the entrance of the peptide into the mitochondria. The ferre-doxin protein acts as a shuttle accepting electrons from ferredoxin oxidoreductase, which then diffuses in the mitochondrial matrix to a P450 hydroxylase (see Figure 2-29) where it donates a pair of electrons.

3. Ferredoxin Oxidoreductase

Ferredoxin oxidoreductase (reaction 15 in Table 26) is an inner mitochondrial membrane-bound flavo-protein with a molecular weight of 51,100. It is responsible for the transfer of electrons from NADPH to ferredoxin and is widely expressed in many human tissues.

4. Cholesterol Side Chain Cleavage Enzyme

The two enzymes that catalyze the cleavage of the side chain of cholesterol (3) [to yield pregnenolone (72)] and that of 17a-hydroxypregnenolone (74) [to yield androst-4-ene-3,17-dione (25)] are known as lyases. Lyases are enzymes that cleave bonds between carbons bearing vicinal hydroxyls. The properties of these two reactions will be discussed separately.

The cholesterol side chain cleavage enzyme (P450scc) (reaction 1 in Table 2-6) carries out three successive enzymatic steps. These involve the 20R- and 22R-hydroxylations followed by cleavage of the side chain on the two carbons bearing the newly incorporated hydroxyl group. Each of these steps is an oxidation-reduction reaction requiring a pair of electrons, which is supplied by NADPH. The P450scc has been cloned and expressed. The protein subunit molecular weight is 49,000, but the functional P450scc contains 16 subunits, creating a multimer of 850,000 that is associated with the inner mitochondrial membrane. The P450scc from the human adrenal and testes have identical sequences.

5. 17a-Hydroxylase/C-l 7-C-20 Lyase

Another enzyme that carries out both a hydroxylation reaction and cleavage of a carbon-carbon bond is the P450cl7 (reactions 7 and 8 in Table 2-6). The P450cl7 enzymes from both the adrenals and testes

Cholesterol Pregnenolone

FIGURE 2-29 Proposed shuttle role of ferredoxin in mitochondrial P450 hydroxylases. The nonheme iron ferredoxin (Adx+), which is freely diffusible in the mitochondrial matrix, accepts a pair of electrons from ferredoxin oxidoreductase (AdRed). After diffusion, the ferredoxin donates a pair of electrons to a P450 (P450scc in this model). The uncharged ferredoxin then diffuses back to the ferredoxin oxidoreductase. Modified with permission from Miller, W. (1988). Molecular biology of steroid hormone synthesis. Endocr. Rev. 9, 295-318.

Cholesterol Pregnenolone

FIGURE 2-29 Proposed shuttle role of ferredoxin in mitochondrial P450 hydroxylases. The nonheme iron ferredoxin (Adx+), which is freely diffusible in the mitochondrial matrix, accepts a pair of electrons from ferredoxin oxidoreductase (AdRed). After diffusion, the ferredoxin donates a pair of electrons to a P450 (P450scc in this model). The uncharged ferredoxin then diffuses back to the ferredoxin oxidoreductase. Modified with permission from Miller, W. (1988). Molecular biology of steroid hormone synthesis. Endocr. Rev. 9, 295-318.

have been cloned and expressed and found to be identical proteins. The enzyme P450cl7 in the adrenals is involved in the production of Cortisol (13) (see Figure 2-21), which has a 17a-hydroxyl, while the P450cl7 in the testes is involved in the production of testosterone (15) and androst-5-ene-3/3,17-diol (85) (see Figure 222). This suggests the existence of a cellular regulatory mechanism in the adrenals to suppress the P450cl7 catalytic ability for C-17-C-20 side chain cleavage.

6. llfi-Hydroxylase

The P450cll is still another example of one enzyme that can catalyze more than one enzymatic reaction; this single P450 has 11/3-hydroxylase, 18-hydroxylase, and 18-oxidase activities (reactions 4-6 in Table 2-6). Highly purified P450cll (molecular weight 48,000) from bovine adrenal zona glomerulosa and zona fasci-culata mitochondria displayed all three enzyme activities and were indistinguishable enzymatically, by amino acid composition, and immunologically. But intact mitochondria from the adrenal zona fasciculata lacks aldehyde synthetase activity, which suggests the presence of cell-specific factors that suppress the 18-hydroxylase activity. In addition, it is known that there are at least two genes for P450cll; one gene codes for the functional 11/3-hydroxylase in the zona reticularis, while the second gene codes for the aldosterone synthesis capability of the zona glomerulosa. The existence of two genes is consistent with known human clinical deficiency states associated with congenital adrenal hyperplasia (see Chapter 10).

7. 21-Hydroxylase

The 21-hydroxylation essential to the production of Cortisol (reaction 3 in Table 2-6) is mediated by P450c21, which is found in the endoplasmic reticulum. Evidence has been presented that humans have two genes coding for P450c21. The details of the expression of these genes are clinically important since about 1 in every 7000 individuals has an autosomally recessive genetic lesion in this enzyme. A detailed discussion of this topic is beyond the scope of this chapter.

8. Aromatase

The aromatization of the A ring of either testosterone (15) to yield estradiol (16) or androst-4-ene-3,17-dione (25) to yield estrone (88) (see Figure 2-23 and reaction 10 of Table 2-6) is mediated by P450aro. This enzyme is present in the endoplasmic reticulum of the ovary and placenta and also occurs to a limited extent in brain and adipose tissues. Figure 2-30 presents a proposed mechanism for the complex aromatization process. To date, two genes for human P450aro have been detected.

9. Ad4-Binding Protein

One of the features of the tissue expression of P450 steroid-metabolizing enzymes is their specific appearance in only steroidogenic tissues (see Table 2-5). Evi-

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