H2o

h crotonyl ACP

Acyl carrier protein crotonyl ACP

h3c ch2\

butyryl ACP

Acyl carrier protein

Acyl carrier protein

Figure 3.5 The first and second round of steps in the synthesis of a fatty acid in animals.

These stages are repeated until the chain reaches its full length of 16 or 18 carbon atoms of the fatty acids. B~ is a basic group on the enzyme

Figure 3.5 The first and second round of steps in the synthesis of a fatty acid in animals.

These stages are repeated until the chain reaches its full length of 16 or 18 carbon atoms of the fatty acids. B~ is a basic group on the enzyme bond is formed in degradation by the use of FAD, and that NADPH is used for the reduction of C=0 to CHOH in synthesis, while NAD+ (without the extra phosphate) is used in the oxidation of CHOH to C=0. In synthesis an (i?)-OH group is formed by reduction, while in degradation an (S)-OH is the intermediate by hydration of the double bond (Figure 3.3). There is also spatial separation of these processes, synthesis occurs in the cytosol, the liquid portion of the cytoplasm, while degradation takes place in the mitochrondria. These are further examples of the way that degradation and synthesis are separated as chemical processes.

3.1.2 Unsaturated Acids and Desaturase Enzymes

Double bonds, as in oleic acid (Figure 3.1), can be introduced in two ways, either a double bond is left in the growing chain (in anaerobic bacteria) or by removal of two hydrogen atoms from the complete molecule (aerobically, in plants and animals, including insects). This latter is a most remarkable reaction and worth a closer examination. It also illustrates the action of a type of biosynthetic enzyme that has received a great deal of investigation. Double bonds are introduced by desaturase enzymes. They are remarkable because they can remove hydrogen atoms from an unactivated alkyl chain, with great precision of position and stereochemistry, something that chemists have not yet learned to do. The C-H bond is very stable, it has a bond enthalpy of 413 kJ mol-1 or 98.7 kcal mol-1. It requires the oxidative power of 02 to break this bond. The byproduct is water.

There are two types of desaturase; the first are soluble enzymes, found only in plants, and located in the plastids. Their substrate is a fatty acid attached to acyl carrier protein (acyl ACP). The second type is integrally bound to the membrane of the endoplasmic reticulum. These enzymes are found in animals generally, including insects, as well as in plants and fungi. Their substrate is the fatty acid bound to coenzyme A (acyl CoA).

Soluble enzymes are much easier to study, so more is known of the first type, but from many studies with a variety of spectroscopic, X-ray and molecular biological techniques, it seems the mechanism of reaction is the same in both types. Although the full story of the enzymes is not yet known, the description here summarizes our present knowledge of membrane-bound fatty acid desaturases found in insects. The description is of a A9-desaturase, the most common type, which converts stearic acid to oleic acid. The location of the double bond is measured from the carboxylate end of the molecule. Palmitic acid with the same enzyme gives palmitoleic acid. If an unnatural C17 or C19 acid is supplied to the desaturase enzyme, a A-9 acid is always formed. The double bonds introduced in this way in fatty acids always have a Z or cis configuration. There are other enzymes that produce a double bond at different positions in the chain, and with different geometry. Desaturase enzymes of insects usually place double bonds at uneven positions in the fatty acids.

The desaturase protein chain consists essentially of four a-helix coils, imbedded in the membrane, with a long narrow pocket into which the alkyl end of the fatty acid fits. The general appearance is shown for a soluble A9-desaturase from the castor oil plant in Figure 3.6 and Plate 1. Note the bent configuration that the molecule assumes at the C-9 atom (regardless of chain length), where the new double bond will be formed, and the two iron atoms within catalytic distance of this position. The iron atoms are buried deep inside the molecule, held by several histidine residues. When the substrate fits into the pocket, oxygen becomes attached to the di-iron cluster (Figure 3.7), which then attacks the hydrocarbon chain (Figure 3.8). The subsequent steps are still not known in great detail.

Two further enzymes, both bound in the same membrane, and two coenzymes are required to complete the cycle. Cytochrome b5 restores the desaturase iron to its reduced state, and the cytochrome in turn is reconverted to its reduced state by cytochrome b5 reductase which uses FAD as coenzyme. The FAD is converted to FADH2 (see Chapter 2) by NADPH.

Small variations in the enzyme structure give other desaturases,

Region involved in regiospecificty

Hydrophobi substrate cavity

Region involve in chain length specificity

Diiron active site

Figure 3.6 A drawing of the A9-desaturase enzyme from the castor oil plant showing the monomer with the long, narrow pocket into which the fatty acid, shown as a curved line, fits. Note the di-iron cluster, and the region at the bottom of the cavity which determines how deep the fatty acid can fit, and consequently where the double bond will be inserted. The direction from which the electrons come from cytochrome which re-oxidize the iron cluster is also shown. See also Plate 1. Figure provided by J. Shanklin and E. Cahoon

Diiron active site

Possible electron transport routes Fd e

Figure 3.6 A drawing of the A9-desaturase enzyme from the castor oil plant showing the monomer with the long, narrow pocket into which the fatty acid, shown as a curved line, fits. Note the di-iron cluster, and the region at the bottom of the cavity which determines how deep the fatty acid can fit, and consequently where the double bond will be inserted. The direction from which the electrons come from cytochrome which re-oxidize the iron cluster is also shown. See also Plate 1. Figure provided by J. Shanklin and E. Cahoon

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