Palmitate fatty acid synthesis

The cyclic process of fatty acid synthesis may be represented by a series of consecutive reactions (hereafter palmitate synthetase is... 

The key enzymes involved in the biosynthetic pathways of the Type I compounds are the fatty acid synthesis enzymes acetyl-CoA carboxylase and fatty acid synthetase. These enzymes are similar to those that produce the normal fatty acids used by all organisms. The resulting products are palmitic (16 car-... 

FIGURE 3-7 Pathways for the interconversion of brain fatty acids. Palmitic acid (16 0) is the main end product of brain fatty acid synthesis. It may then be elongated, desaturated, and/or P-oxidized to form different long chain fatty acids. The monoenes (18 1 A7, 18 1 A9, 24 1 A15) are the main unsaturated fatty acids formed de novo by A9 desaturation and chain elongation. As shown, the very long chain fatty acids are a-oxidized to form a-hydroxy and odd numbered fatty acids. The polyunsaturated fatty acids are formed mainly from exogenous dietary fatty acids, such as linoleic (18 2, n-6) and a-linoleic (18 2, n-3) acids by chain elongation and desaturation at A5 and A6, as shown. A A4 desaturase has also been proposed, but its existence has been questioned. Instead, it has been shown that unsaturation at the A4 position is effected by retroconversion i.e. A6 unsaturation in the endoplasmic reticulum, followed by one cycle of P-oxidation (-C2) in peroxisomes [11], This is illustrated in the biosynthesis of DHA (22 6, n-3) above. In severe essential fatty acid deficiency, the abnormal polyenes, such as 20 3, n-9 are also synthesized de novo to substitute for the normal polyunsaturated acids. 

By 1960 it was clear that acetyl CoA provided its two carbon atoms to the to and co—1 positions of palmitate. All the other carbon atoms entered via malonyl CoA (Wakil and Ganguly, 1959 Brady et al. 1960). It was also known that 3H-NADPH donated tritium to palmitate. It had been shown too that fatty acid synthesis was very susceptible to inhibition by p-hydroxy mercuribenzoate, TV-ethyl maleimide, and other thiol reagents. If the system was pre-incubated with acetyl CoA, considerable protection was afforded against the mercuribenzoate. In 1961 Lynen and Tada suggested tightly bound acyl-S-enzyme complexes were intermediates in fatty acid synthesis in the yeast system. The malonyl-S-enzyme complex condensed with acyl CoA and the B-keto-product reduced by NADPH, dehydrated, and reduced again to yield the (acyl+2C)-S-enzyme complex. Lynen and Tada thought the reactions were catalyzed by a multifunctional enzyme system. 

Figure 11.6 The physiological pathway for fatty acid synthesis acetyl-CoA to palmitoyl-CoA. The pathway starts with the conversion of acetyl-CoA to malonyl-CoA in the cytosol, which is the flux-generating step catalysed by acetyl-CoA carboxylase. The pathway can be considered to end with formation of palmitoyl-CoA rather than palmitate, since it has several fates formation of triacylglycerol and phospholipids or acylation of other compounds.

Net Equation of Fatty Acid Synthesis Write the net equation for the biosynthesis of palmitate in rat liver, starting from mitochondrial acetyl-CoA and cytosolic NADPH, ATP, and C02. 

The pathway The first committed step in fatty acid biosynthesis is the carboxylation of acetyl CoA to form malonyl CoA which is catalyzed by the biotin-containing enzyme acetyl CoA carboxylase. Acetyl CoA and malonyl CoA are then converted into their ACP derivatives. The elongation cycle in fatty acid synthesis involves four reactions condensation of acetyl-ACP and malonyl-ACP to form acetoacetyl-ACP releasing free ACP and C02, then reduction by NADPH to form D-3-hydroxybutyryl-ACP, followed by dehydration to crotonyl-ACP, and finally reduction by NADPH to form butyryl-ACP. Further rounds of elongation add more two-carbon units from malonyl-ACP on to the growing hydrocarbon chain, until the C16 palmitate is formed. Further elongation of fatty acids takes place on the cytosolic surface of the smooth endoplasmic reticulum (SER). 

The rate limiting step in fatty acid synthesis is catalyzed by acetyl-CoA carboxylase to produce malonyl-CoA at the expense of one ATP.31 Malonate and acetate are transferred from CoA to acyl carrier protein in the cytosolic fatty acid synthetase complex, where chain extension leads to the production of palmitate. Palmitate can then be transferred back to CoA, and the chain can be extended two carbons at a time through the action of a fatty acid elongase system located in the endoplasmic reticulum. The >-hydroxylation that produces the >-hydroxyacids of the acylceramides is thought to be mediated by a cytochrome p450 just when the fatty acid is long enough to span the endoplasmic reticular membrane. 

Note that fatty acid synthesis provides an extreme example of the phenomenon of metabolic channeling neither free fatty acids with more than four carbons nor their CoA derivatives can directly participate in the synthesis of palmitate. Instead they must be broken down to acetyl-CoA and reincorporated into the fatty acid. 

Figure 19.13 Biosynthesis of palmitate via fatty acid synthetase. The numbered enzyme activities (steps) are as follows (1) acetyl-CoA transacylase (2) malonyl-coA transacylase (3) /3-ketoacylsynthetase (4) j3-ketoacylreductase (5) /3-hydroxyacyldehydratase (6) enoyl reductase (7) fatty acyltransacylase. (Reproduced by permission from Wakil SJ, Stoops JK, Joshi VC. Fatty acid synthesis and its regulation. Annu Rev Biochem 52 537-579, 1983.)...

The synthesis of palmitic acid occurs in the cytosol, from acetyl-CoA. When glucose is abundant and the amount of citrate in the mitochondrial matrix exceeds the demand by the citric acid cycle, the excess citrate is transported out of the mitochondria into the cytosol (Fig. 13-8). Citrate in the cytosol is the source of acetyl groups for fatty acid synthesis, and its metabolism there involves the following enzyme reactions ... 

For the conversion of pyruvate to oxaloacetate and the formation of citrate in the mitochondrion, see Chap. 12. Acetyl-CoA for fatty acid synthesis is converted to malonyl-CoA this reaction is catalyzed by acetyl-CoA carboxylase. Seven molecules of acetyl-CoA are converted to malonyl-CoA for the synthesis of one molecule of palmitic acid. 

In the second round of fatty acid synthesis, butyryl ACP condenses with malonyl ACP to form a C5-P-ketoacyl ACP. This reaction is like the one in the first round, in which acetyl ACP condenses with malonyl ACP to form a C4-P-ketoacyl ACP. Reduction, dehydration, and a second reduction convert the C5-P-ketoacyl ACP into a C5-acyl ACP, which is ready for a third round of elongation. The elongation cycles continue until Ci5-acyl ACP is formed. This intermediate is a good substrate for a thioesterase that hydrolyzes C 15-acyl ACP to yield palmitate and ACP. The thioesterase acts as a ruler to determine fatty acid chain length. The synthesis of longer-chain fatty acids is discussed in Section 22.6. 

Tracing carbons. Consider a cell extract that actively synthesizes palmitate. Suppose that a fatty acid synthase in this preparation forms one molecule of palmitate in about 5 minutes. A large amount of malonyl CoA labeled with i C in each carbon of its malonyl unit is suddenly added to this system, and fatty acid synthesis is stopped a minute later by altering the pH. The fatty acids in the supernatant are analyzed for radioactivity. Which carbon atom of the palmitate formed by this system is more radioactive—C-1 or C-14 ... 

Figure 6-7. Fatty acid synthesis. Malonyl CoA provides the 2-carbon units that are added to the growing fatty acyl chain. The addition and reduction steps are repeated until palmitic acid is produced. P = a phosphopantetheinyl group attached to the fatty acid synthase complex Cys-SH = a cys-teinyl residue.

We already mentioned that the enzymes involved in the P-oxidation of fatty acids are located in the mitochondria. The source of two-carbon fragments for the biosynthesis of both fatty acids and isoprenoids like cholesterol is acetyl CoA, which is generated by oxidative metabolism in the mitochondria. Acetyl CoA cannot escape from the mitochondria, but it can be exported to the cyosol as citrate, where it is reconverted to oxaloacete and acetyl CoA. Fatty acid (and cholesterol) biosynthesis takes place in the cyosol, and requires bicarbonate, which is incorporated into acetyl CoA to form malonyl CoA by acetyl CoA carboxylase. The biosynthesis of fatty acids, mostly the Cie palmitate (Chapter 4), requires one molecule of acetyl CoA and seven molecules of malonyl CoA. In animals, the seven enzymatic reactions which are required for fatty acid synthesis are present in a single multifunctional protein complex, known as fatty acid synthase. The synthase also contains an acyl-carrier protein... 

Fatty acid synthesis is also carried out by a multienzyme complex and leads from acetyl-CoA, malonyl-CoA, and NADPH to palmitic acid. The overall process is as follows ... 

The answer is d. (Murray, pp 230-267. Scriver, pp 2297-2326. Sack, pp 121-138. Wihon, pp 287-320.) In humans, the end product of fatty acid synthesis in the cytosol is palmitic acid. The specilicity of cytosolic multienzyme, single-protein fatty acid synthetase is such that once the C16 chain length is reached, a thioesterase clips off the fatty acid. Elongation as well as desaturation of de novo palmitate and fatty acids obtained from the diet occur by the action of enzymes in the membranes of the endoplasmic reticulum. 

As can be seen from the equations above, the necessary amount of malonyl CoA is synthesized. Palmitate is subsequently synthesized from malonyl CoA and one initial acetyl CoA. Thus, acetyl CoA, NADPH, ATP, and HCOs are all necessary in this process. In contrast, FADH, is not utilized in fatty acid synthesis, but is one of the products of fatty acid oxidation. Vitamin is required for conversion of propionic acid to methylmalonic acid, a step in the p oxidation of odd-numbered fatty acid chains. 

The first step in de novo fatty acid synthesis is the production of malonyl-CoA from acetyl-CoA and bicarbonate. This committed step is catalyzed by acetyl-CoA carboxylase present in the cytoplasm of liver cells and adipocytes. After replacement of the CoA residue in acetyl-CoA by ACP (acyl carrier protein), malonyl-ACP is used to convert acetyl-ACP to butyryl-ACP by the fatty acid synthase complex. In this multistep reaction, NADPH is used as donor of hydrogen atoms and CO2 is produced. Butyryl-ACP is subsequently elongated to hexanoyl-ACP by a similar process in which malonyl-ACP serves as donor of two carbon atoms required for lengthening of the growing acyl chain. This process is repeated until palmitic acid... 

Although fatty acid synthesis occurs within the cytoplasm of most animal cells, liver is the major site for this process. (Recall, for example, that liver produces VLDL. See p. 349.) Fatty acids are synthesized when the diet is low in fat and/or high in carbohydrate or protein. Most fatty acids are synthesized from dietary glucose. As discussed, glucose is converted to pyruvate in the cytoplasm. After entering the mitochondrion, pyruvate is converted to acetyl-CoA, which condenses with oxaloacetate, a citric acid cycle intermediate, to form citrate. When mitochondrial citrate levels are sufficiently high (i.e., cellular energy requirements are low), citrate enters the cytoplasm, where it is cleaved to form acetyl-CoA and oxaloacetate. The net reaction for the synthesis of palmitic acid from acetyl-CoA is as follows ... 

The remaining reactions in fatty acid synthesis take place on the fatty acid synthase multienzyme complex. This complex, the site of seven enzyme activities and ACP, is a 500-kD dimer. Because the enormous polypeptides in the dimer are arranged in a head-to-tail configuration, two fatty acids can be constructed simultaneously. A proposed mechanism for palmitate synthesis is shown in Figure 12.13. 

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