Palmitoyl elongation

The fatty acid synthesis pathway can be seen to occur in two parts. An initial priming stage in which acetyl-CoA is converted to malonyl-CoA by a carboxylation reaction (Figure 6.9) is followed by a series of reactions which occur on a multi-enzyme complex (MEC), which achieves chain elongation forming C16 palmitoyl-CoA. The whole process occurs in the cytosol. 

After each sequence of reactions the acyl group (C4 after one cycle, C6 after two cycles, C8 after three cycles, etc.) re-enters the process at step 3 and undergoes condensation with the next malonyl-CoA. When the final C16 palmitate molecule has been synthesized it is released but needs to be re-activated with CoA, to palmitoyl-CoA for desaturation, elongation into C18 fatty acid molecule or use for triglyceride synthesis (see Figures 6.12, 6.13 and 6.17). 

The FAS multi-enzyme complex synthesizes saturated C16 fatty acids, but cells and tissues need unsaturated and longer chain fatty acids. The palmitoyl-CoA can be modified by either chain elongation and/or oxidation in order to produce different fatty acid molecules. Both elongation and desaturation occur within the smooth endoplasmic reticulum (SER, microsomal fraction) of the cell. 

For the synthesis of a small library of palmitoylated and isoprenylated N-Ras peptides in solution, a modular strategy was adopted, with the tetrapeptide MGLP 38 as a key intermediate. This tetrapeptide allowed further elongation at its C-terminus with lipidated or nonlipidated cysteine methyl esters, as well as the addition of various N-terminally MIC-labeled dipeptides, consisting of different GC lipidated units 39—41... 

Afterward, the peptide chain was elongated following the standard Fmoc-based protocol. Before cleavage of the peptide the incorporated Mmt-protected cysteine was deprotected using 1% TFA. Under these very mild conditions the farnesyl moiety was not harmed. Palmitoylation could he achieved using an excess of palmitoyl chloride. Cleavage with copper acetate and methanol as a nucleophile gave the farnesylated and palmitoylated N-Ras sequence with the C-terminal methyl ester 78. ... 

The key enzyme in fatty acid synthesis is acetyl CoA carboxylase (see p. 162), which precedes the synthase and supplies the malonyl-CoA required for elongation. Like all carboxylases, the enzyme contains covalently bound biotin as a prosthetic group and is hormone-dependently inactivated by phosphorylation or activated by dephosphorylation (see p. 120). The precursor citrate (see p. 138) is an allosteric activator, while palmitoyl-CoA inhibits the end product of the synthesis pathway. 

Seven cycles of condensation and reduction produce the 16-carbon saturated palmitoyl group, still bound to ACP. For reasons not well understood, chain elongation by the synthase complex generally stops at this point and free palmitate is released from the ACP by a hydrolytic activity in the complex. Small amounts of longer fatty acids such as stearate (18 0) are also formed. In certain plants (coconut and palm, for example) chain termination occurs earlier up to 90% of the fatty acids in the oils of these plants are between 8 and 14 carbons long. 

Palmitoyl can be further elongated in the endoplasmic reticulum and the mitochondria, and desaturated by mixed function oxidases in the endoplasmic reticulum. 

The (3 subunit of the Py complex of transducin is a seven-bladed (3 propeller (Fig.ll-7C). It is composed of seven GH-WD repeat units - [GH - Xn - WD]4 8-where GH = Gly - His, WD = Trp-Asp, and Xn is a core repeating sequence, usually 32 -42 residues in length. This motif is also found in at least 40 other eukaryotic proteins.188/237 -240 In the Py complex (Fig. 11-7B) the y subunit assumes an elongated, largely a-helical structure. It is often anchored at its C terminus by a famesyl or geranylgeranyl chain, while Ga may be myristoylated or palmitoylated.195/230/243... 

Enzyme complexes occur in the endoplasmic reticulum of animal cells that desaturate at A5 if there is a double bond at the A8 position, or at A6 if there is a double bond at the A9 position. These enzymes are different from each other and from the A9-desaturase discussed in the previous section, but the A5 and A6 desaturases do appear to utilize the same cytochrome b5 reductase and cytochrome b5 mentioned previously. Also present in the endoplasmic reticulum are enzymes that elongate saturated and unsaturated fatty acids by two carbons. As in the biosynthesis of palmitic acid, the fatty acid elongation system uses malonyl-CoA as a donor of the two-carbon unit. A combination of the desaturation and elongation enzymes allows for the biosynthesis of arachidonic acid and docosahexaenoic acid in the mammalian liver. As an example, the pathway by which linoleic acid is converted to arachidonic acid is shown in figure 18.17. Interestingly, cats are unable to synthesize arachidonic acid from linoleic acid. This may be why cats are carnivores and depend on other animals to make arachidonic acid for them. Also note that the elongation system in the endoplasmic reticulum is important for the conversion of palmitoyl-CoA to stearoyl-CoA. 

This first round of elongation produces the four-carbon butyryl-ACP. The cycle now repeats with malonyl-ACP adding two-carbon units in each cycle to the lengthening acyl-ACP chain. This continues until the 16-carbon palmitoyl-ACP is formed. This molecule is not accepted by the acyl-malonyl-ACP condensing enzyme, and so cannot be elongated further by this process. Instead it is hydrolyzed by a thioesterase to give palmitate and ACP. 

The cycle of transfer, elongation, reduction, dehydration, and reduction continues until palmitoyl-ACP is made. Then the thioesterase activity of the FAS complex releases the 16-carbon fatty acid palmi-tate from the FAS. 

The ER elongation system works similarly to the fatty acid synthetase sequence, except that individual enzymes (gene products) are involved palmitoyl-CoA reacts with malonyl-CoA to give C02 and /3-ketooctadecanoyl-CoA. The... 

If the Fatty Acid Synthetase Complex only makes palmitate where do the rest of the fatty acids come from Of course palmitate can be shortened by P-oxidation. For longer fatty acids there is a fatty acid elongation system localized on the ER. The same reactions occur as in the S)mthetase, but now have individual enzymes. Palmitate is first activated to palmitoyl-CoA. The enzymes prefer C-16 or less as... 

One molecule of oxygen accepts two pairs of electrons, one from palmitoyl-CoA and the other from NADPH or NADH. The electrons NAD(P)H are transported via cytochrome-bs reductase to cytochrome bs (microsomal electron transport Chapter 14). An enzyme-bound superoxide radical is responsible for the oxidation of acyl-CoA. Four desaturases specific for introducing cis double bonds at C9, Ca, C5, and C4, respectively, are known. If the substrate is saturated, the first double bond introduced is C9. With an unsaturated substrate, other double bonds are introduced between the carboxyl group and the double bond nearest the carboxyl group. Desaturation yields a divinylmethane arrangement of double bonds (—CH=CH—CH2—CH=CH—). Usually desaturation alternates with chain elongation. Desaturation is inhibited by fasting and diabetes. The oxidation of unsaturated fatty acids occurs in mitochondria. 

As shown in Figure 22.22 on page 619 of the text, acetyl-ACP and malonyl-ACP condense to form acetoacetyl-ACP. Carbons 4 and 3 of acetoacetyl-ACP are not labeled, because they are derived from acetyl CoA. These two carbons vdll become carbons 15 and 16 of palmitate. Only C-2 of acetoacetyl-ACP will be labeled because it is derived from the methylene carbon of malonyl-ACP. When the second round of synthesis begins, butyryl-ACP condenses with a second molecule of methylene-labeled malonyl-ACP, which contributes C-1 and C-2 of the newly formed six-carbon ACP derivative. In this compound, C-2 and C-4 will be labeled. Chain elongation continues until palmitoyl-ACP is formed. Each even-numbered carbon atom, except for carbon 16 (at the co end), will be labeled. 

Fig. 4. The effect of increasing concentrations of bovine serum albumin on the rate of condensation ( ) and overall chain elongation (o) using palmitoyl-CoA as substrate. From Bernert and Sprecher (1979b).

The factors altering chain elongation activity have not been as carefully defined as they have for the various desaturases. Fasting does depress the rate of chain elongation of palmitoyl-CoA to stearoyl-CoA and activity is... 

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