Palmitate and stearate serve as precursors of the two most common monounsaturated fatty acids of animal tissues: palmitoleate, 16:1(Δ9), and oleate, 18:1(Δ9) (Fig. 20-13). Each of these fatty acids has a single cis double bond in the Δ9 position (between C-9 and C-10). The double bond is introduced into the fatty acid chain by an oxidative reaction catalyzed by fatty acyl-CoA desaturase (Fig. 20-14). This enzymeis an example of a mixed-function oxidase (Box 20-1). Two different substrates, the fatty acid and NADPH, simultaneously undergo twoelectron oxidations. The path of electron flow includes a cytochrome (cytochrome b5) and a flavoprotein (cytochrome b5 reductase), both of which, like fatty acyl-CoA desaturase itself, are present in the smooth endoplasmic reticulum.
Mammalian hepatocytes can readily introduce double bonds at the Δ9 position of fatty acids but cannot introduce additional double bonds in the fatty acid chain between C-10 and the methyl-terminal end.
Figure 20-14 The pathway of electron transfer (blue arrows) in the desaturation of fatty acids by a mixed-function oxidase in vertebrate animals. 'hvo different substrates-a fatty acyl-CoA and NADPH-undergo oxidation by molecular oxygen. These reactions occur on the lumenal face of the smooth endoplasmic reticulum. A similar pathway, but with difierent electron carriers, occurs in plants.
Linoleate, 18:2(Δ9,12), and α-linolenate, 18:3(Δ9,12,15, cannot be synthesized by mammals, but plants can synthesize both. The plant desaturases that introduce double bonds at Δ12 and Δ15 positions are located in the endoplasmic reticulum. These enzymes act not on free fatty acids but on a phospholipid, phosphatidylcholine, containing at least one oleate linked to glycerol (Fig. 20-15, p. 656).
Because they are necessary precursors for the synthesis of other products, linoleate and linolenate are essential fatty acids for mammals; they must be obtained from plant material in the diet. Once ingested, linoleate may be converted into certain other polyunsaturated acids, particularly y-linolenate, eicosatrienoate, and eicosatetraenoate (arachidonate), which can be made only from linoleate (Fig. 20-13). Arachidonate, 20: 4(Δ5,8,11,14), is an essential precursor of regulatory lipids, the eicosanoids.
B O X 20-1 Mixed-Function Oxidases, Oxygenases, and Cytochrome P-450In this chapter we encounter several enzymes that carry out oxidation-reduction reactions in which molecular oxygen is a participant. The reaction that introduces a double bond into a fatty acyl chain (Fig. 20-14) is such a reaction. The nomenclature for enzymes that catalyze reactions of this general type is often confusing to students, as is the mechanism. Oxidase is the general name for enzymes that catalyze oxidations in which molecular oxygen is the electron acceptor but oxygen atoms do not appear in the oxidized product (but there is an exception to this "rule," as we shall see!). The enzyme that creates a double bond in fatty acyl-CoA during the oxidation of fatty acids in peroxisomes (see Fig. 16-13) is an oxidase of this type, as is the cytochrome oxidase of the mitochondrial electron transfer chain (see Fig. 18-11). In the first case, the transfer of two electrons to H2O produces hydrogen peroxide, H2O2; in the second, two electrons reduce ½O2 to H2O. Many, but not all, oxidases are flavoproteins. Oxygenases catalyze oxidative reactions in which oxygen atoms are directly incorporated into the substrate molecule, forming a new hydroxyl or carboxyl group, for example. Dioxygenases catalyze reactions in which both of the oxygen atoms of O2 are incorporated into the organic substrate molecule. An example of a dioxygenase is tryptophan 2,3-dioxygenase, which catalyzes the opening of the five-membered ring of tryptophan in the catabolism of this amino acid:
When this reaction occurs in the presence of 18O2, the isotopic oxygen atoms are found in the two carbonyl groups of the product (shown in red). Monooxygenases, which are more abundant and more complex in their action, catalyze reactions in which only one of the two oxygen atoms of 02 is incorporated into the organic substrate, the other being reduced to H2O. Monooxygenases require two substrates to serve as reductants of the two oxygen atoms of O2. The main substrate accepts one of the two oxygen atoms, and a cosubstrate furnishes hydrogen atoms to reduce the other oxygen atom to H2O. The general reaction equation for monooxygenases is AH + BH2 + O-O where AH is the main substrate and BHz the cosubstrate. Because most monooxygenases catalyze reactions in which the main substrate becomes hydroxylated, they are also called hydroxylases. They are also sometimes called mixed-function oxidases or mixed-function oxygenases, to indicate that they oxidize two dif ferent substrates simultaneously. (Note here the use of "oxidase"-a deviation from the general meaning of this term.) There are different classes of monooxygenases, depending upon the nature of the cosubstrate. Some use reduced flavin nucleotides (FMNH2 or FADH2), others use NADH or NADPH, and still others use α-ketoglutarate as the cosubstrate. The enzyme that hydroxylates the phenyl ring of phenylalanine to give tyrosine is a monooxygenase for which tetrahydrobiopterin serves as cosubstrate (see Fig. 17-27). (This is the enzyme that is defective in the human genetic disease phenylketonuria.) The most numerous and most complex monooxygenation reactions are those employing a type of heme protein called cytochrome P-450. This type of cytochrome is usually present in the smooth endoplasmic reticulum rather than the mitochondria. Like mitochondrial cytochrome oxidase, cytochrome P-450 can react with O2 and with carbon monoxide, but it can be differentiated from cytochrome oxidase because the carbon monoxide complex of its reduced form absorbs light strongly at 450 nm; thus the name P-450. Cytochrome P-450 catalyzes hydroxylation reactions in which an organic substrate RH is hydroxylated to R-OH at the expense of one oxygen atom of O2; the other oxygen atom is reduced to H2O by reducing equivalents furnished by NADH or NADPH but usually passed to P-450 by an ironsulfur protein. Figure 1 shows a simplified outline of the action of cytochrome P-450, which has intermediate steps not yet fully understood. Cytochrome P-450 actually is a family of closely similar proteins; serevalhundred members of this protein family are known, each with a different substrate specificity. In the adrenal cortex a specific cytochrome P-450 participates in the hydroxylation of steroids to yield the adrenocortical hormones, for example (Fig. 20-42). Cytochrome P-450 is also important in the hydroxylation of many different drugs, such as barbiturates, and other xenobiotics (substances that are foreign to the body), particularly if they are hydrophobic and relatively insoluble. The environmental carcinogen benzo[α]pyrene (found in cigarette smoke) undergoes cytochrome P-450-dependent hydroxylation in the course of its detoxification. Hydroxylation of such foreign compounds makes them more soluble in water and allows their excretion in the urine. Unfortunately, hydroxylation of some compounds converts them into toxic substances, subverting the detoxification system. Reactions described in this chapter that are catalyzed by mixed-function oxidases are those involved in fatty acyl-CoA desaturation (Fig. 20-14); leukotriene synthesis (Fig. 20-17), plasmalogen synthesis (Fig. 20-28), conversion of squalene to cholesterol (Fig. 20-35), and steroid hormone synthesis (Fig. 20-42).
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Arachidonate is parent to the eicosanoids, a family of very potent biological signaling molecules that act as short-range messengers, affecting tissues near the cells that produce them. In response to a hormonal or other stimulus, a specific phospholipase present in most types of mammalian cells attacks membrane phospholipids, releasing arachidonate. Enzymes of the smooth endoplasmic reticulum then convert arachidonate into prostaglandins, beginning with the formation of PGH2, the immediate precursor of many other prostaglandins and thromboxanes (Fig. 20-16a). The two reactions that lead to PGH2 involve the addition of molecular oxygen; both are catalyzed by a bifunctional enzyme, prostaglandin endoperoxide synthase. Aspirin (acetylsalicylate; Fig. 20-16b) irreversibly inactivates this enzyme (thus blocking the synthesis of prostaglandins and thromboxanes) by acetylating a Ser residue essential to catalytic activity. Ibuprofen, a widely used nonsteroidal antiinflammatory drug (Fig. 20-16c), also acts by inhibiting this enzyme. Figure 20-15 Desaturases in plants oxidize phosphatidylcholine-bound oleate, producing polyunsaturated fatty acids. Some of the products are released from phosphatidylcholine by hydrolysis. |
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Figure 20-16 The "cyclic" pathway from arachidonate to prostaglandins and thromboxanes. (a) After release of arachidonate, prostaglandin endoperoxide synthase catalyzes the first two reactions, producing PGH2, the precursor of other prostaglandins and thromboxanes. (b) Aspirin inhibits prostaglandin endoperoxide synthase by acetylating an essential Ser residue on the enzyme. Ibuprofen (c) also inhibits this step, probably by mimicking the structure of the substrate or an intermediate in the reaction.
Thromboxane synthase present in blood platelets (thrombocytes) converts PGH2 into thromboxane A2, from which other thromboxanes are derived (Fig. 20-16a). Thromboxanes induce blood vessel constriction and platelet aggregation, early steps in blood clotting. Low doses of aspirin, taken regularly, are believed to reduce the probability of heart attacks and strokes by reducing thromboxane production.
Thromboxanes, like prostaglandins, contain a ring of five or six atoms, and the pathway that leads from arachidonate to these two classes of compounds is sometimes called the "cyclic" pathway, to distinguish it from the "linear" pathway that leads from arachidonate to the leukotrienes, which are linear (Fig. 20-17, p. 658). Leukotriene synthesis begins with the action of several lipoxygenases that catalyze the incorporation of molecular oxygen into arachidonate. These enzymes, found in leukocytes and in heart, brain, lung, and spleen, are mixed-function oxidases that use cytochrome P-450 (Box 20-1). The various leukotrienes differ in the position of the peroxide that is introduced by these lipoxygenases. This linear pathway from arachidonate, unlike the cyclic pathway, is not inhibited by aspirin or the other nonsteroidal antiinflammatory drugs.
A-OH + B + H2O
