Biosynthesis of Fatty Acids

Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc

BIOMEDICAL IMPORTANCE

Fatty acids are synthesized by an extramitochondrial system, which is responsible for the complete synthesis of palmitate from acetyl-CoA in the cytosol. In the rat, the pathway is well represented in adipose tissue and liver, whereas in humans adipose tissue may not be an important site, and liver has only low activity. In birds, lipogenesis is confined to the liver, where it is particularly important in providing lipids for egg formation. In most mammals, glucose is the primary substrate for lipogenesis, but in ruminants it is acetate, the main fuel molecule produced by the diet. Critical diseases of the pathway have not been reported in humans. However, inhibition of lipogenesis occurs in type 1 (insulin-dependent) diabetes mellitus, and variations in its activity may affect the nature and extent of obesity.

THE MAIN PATHWAY FOR DE NOVO SYNTHESIS OF FATTY ACIDS (LIPOGENESIS) OCCURS IN THE CYTOSOL

This system is present in many tissues, including liver, kidney, brain, lung, mammary gland, and adipose tissue. Its cofactor requirements include NADPH, ATP, Mn2+, biotin, and HCO3- (as a source of CO2). Acetyl-CoA is the immediate substrate, and free palmitate is the end product.

Production of Malonyl-CoA Is the Initial & Controlling Step in Fatty Acid Synthesis

Bicarbonate as a source of CO2 is required in the initial reaction for the carboxylation of acetyl-CoA to mal-onyl-CoA in the presence of ATP and acetyl-CoA car-boxylase. Acetyl-CoA carboxylase has a requirement for the vitamin biotin (Figure 21-1). The enzyme is a multienzyme protein containing a variable number of identical subunits, each containing biotin, biotin car-boxylase, biotin carboxyl carrier protein, and transcar-boxylase, as well as a regulatory allosteric site. The reaction takes place in two steps: (1) carboxylation of biotin involving ATP and (2) transfer of the carboxyl to acetyl-CoA to form malonyl-CoA.

The Fatty Acid Synthase Complex Is a Polypeptide Containing Seven Enzyme Activities

In bacteria and plants, the individual enzymes of the fatty acid synthase system are separate, and the acyl radicals are found in combination with a protein called the acyl carrier protein (ACP). However, in yeast, mammals, and birds, the synthase system is a multienzyme polypeptide complex that incorporates ACP, which takes over the role of CoA. It contains the vitamin pantothenic acid in the form of 4'-phosphopan-tetheine (Figure 45-18). The use of one multienzyme functional unit has the advantages of achieving the effect of compartmentalization of the process within the cell without the erection of permeability barriers, and synthesis of all enzymes in the complex is coordinated since it is encoded by a single gene.

In mammals, the fatty acid synthase complex is a dimer comprising two identical monomers, each containing all seven enzyme activities of fatty acid synthase on one polypeptide chain (Figure 21-2). Initially, a priming molecule of acetyl-CoA combines with a cys-teine —SH group catalyzed by acetyl transacylase (Figure 21-3, reaction 1a). Malonyl-CoA combines with the adjacent —SH on the 4'-phosphopantetheine of ACP of the other monomer, catalyzed by malonyl transacylase (reaction 1b), to form acetyl (acyl)-mal-onyl enzyme. The acetyl group attacks the methylene group of the malonyl residue, catalyzed by 3-ketoacyl synthase, and liberates CO2, forming 3-ketoacyl enzyme (acetoacetyl enzyme) (reaction 2), freeing the cys-teine —SH group. Decarboxylation allows the reaction to go to completion, pulling the whole sequence of reactions in the forward direction. The 3-ketoacyl group is reduced, dehydrated, and reduced again (reactions 3, 4, 5) to form the corresponding saturated acyl-S-enzyme. A new malonyl-CoA molecule combines with the —SH of 4'-phosphopantetheine, displacing the saturated acyl residue onto the free cysteine —SH group. The sequence of reactions is repeated six more times until a saturated 16-carbon acyl radical (palmityl) has been assembled. It is liberated from the enzyme complex by the activity of a seventh enzyme in the complex, thioesterase (deacylase). The free palmitate must be activated to acyl-CoA before it can proceed via any other

CH3— CO r\j S - CoA-—---OOC — CH2— CO r\j S — CoA

Acetyl-CoA S Malonyl-CoA

Figure 21-1. Biosynthesis of malonyl-CoA. (Enz, acetyl-CoA carboxylase.)

metabolic pathway. Its usual fate is esterification into acylglycerols, chain elongation or desaturation, or esterification to cholesteryl ester. In mammary gland, there is a separate thioesterase specific for acyl residues of C8, C10, or C12, which are subsequently found in milk lipids.

The equation for the overall synthesis of palmitate from acetyl-CoA and malonyl-CoA is:

CH2CO • S • CoA + 7HOOC • CH2CO • S • CoA +14NADPH +14H+ ^ CH3(CH2 )14COOH + 7CO2 + 6H2O + 8CoA • SH + 14NADP+

The acetyl-CoA used as a primer forms carbon atoms 15 and 16 of palmitate. The addition of all the subsequent C2 units is via malonyl-CoA. Propionyl-CoA acts as primer for the synthesis of long-chain fatty

The acetyl-CoA used as a primer forms carbon atoms 15 and 16 of palmitate. The addition of all the subsequent C2 units is via malonyl-CoA. Propionyl-CoA acts as primer for the synthesis of long-chain fatty

Palmitate Synthesis

Cys I

division

Cys I

4'-Phospho-2. pantetheine division sH

Cys sH

4'-Phospho-2. pantetheine sH

Fatty Acid Synthase Type And

Figure 21-2. Fatty acid synthase multienzyme complex. The complex is a dimer of two identical polypeptide monomers, 1 and 2, each consisting of seven enzyme activities and the acyl carrier protein (ACP). (Cys—SH, cysteine thiol.) The —SH of the 4'-phosphopantetheine of one monomer is in close proximity to the —SH of the cysteine residue of the ketoacyl synthase of the other monomer, suggesting a "head-to-tail" arrangement of the two monomers. Though each monomer contains all the partial activities of the reaction sequence, the actual functional unit consists of one-half of one monomer interacting with the complementary half of the other. Thus, two acyl chains are produced simultaneously. The sequence of the enzymes in each monomer is based on Wakil.

Figure 21-2. Fatty acid synthase multienzyme complex. The complex is a dimer of two identical polypeptide monomers, 1 and 2, each consisting of seven enzyme activities and the acyl carrier protein (ACP). (Cys—SH, cysteine thiol.) The —SH of the 4'-phosphopantetheine of one monomer is in close proximity to the —SH of the cysteine residue of the ketoacyl synthase of the other monomer, suggesting a "head-to-tail" arrangement of the two monomers. Though each monomer contains all the partial activities of the reaction sequence, the actual functional unit consists of one-half of one monomer interacting with the complementary half of the other. Thus, two acyl chains are produced simultaneously. The sequence of the enzymes in each monomer is based on Wakil.

Acetyl-CoA

Fatty acid synthase multienzyme complex

Acetyl-CoA

NADPH+H+

NADPH+H+

NADP+

THIOESTERASE

After cycling through steps seven times

Palmitate

2 V Pan—S r\j C— CH2 — COO Acyl(acetyl)-malonyl enzyme

3-KETOACYL SYNTHASE

OO Il II

3-Ketoacyl enzyme (acetoacetyl enzyme)

3-KETOACYL REDUCTASE

OH I

Pan—S^jC— CH2 —CH —CH3 D(-)-3-Hydroxyacyl enzyme

HYDRATASE

Pan —S ^^ C— CH = CH —CH3 2,3-Unsaturated acyl enzyme

ENOYL REDUCTASE

O II

individual monomers of fatty acid synthase

Figure 21-3. Biosynthesis of long-chain fatty acids. Details of how addition of a malonyl residue causes the acyl chain to grow by two carbon atoms. (Cys, cysteine residue; Pan, 4'-phosphopante-theine.) The blocks shown in dark blue contain initially a C2 unit derived from acetyl-CoA (as illustrated) and subsequently the Cn unit formed in reaction 5.

acids having an odd number of carbon atoms, found particularly in ruminant fat and milk.

The Main Source of NADPH for Lipogenesis Is the Pentose Phosphate Pathway

NADPH is involved as donor of reducing equivalents in both the reduction of the 3-ketoacyl and of the 2,3-unsaturated acyl derivatives (Figure 21-3, reactions 3 and 5). The oxidative reactions of the pentose phosphate pathway (see Chapter 20) are the chief source of the hydrogen required for the reductive synthesis of fatty acids. Significantly, tissues specializing in active lipogenesis—ie, liver, adipose tissue, and the lactating mammary gland—also possess an active pentose phos phate pathway. Moreover, both metabolic pathways are found in the cytosol of the cell, so there are no membranes or permeability barriers against the transfer of NADPH. Other sources of NADPH include the reaction that converts malate to pyruvate catalyzed by the "malic enzyme" (NADP malate dehydrogenase) (Figure 21-4) and the extramitochondrial isocitrate dehydrogenase reaction (probably not a substantial source, except in ruminants).

Acetyl-CoA Is the Principal Building Block of Fatty Acids

Acetyl-CoA is formed from glucose via the oxidation of pyruvate within the mitochondria. However, it does not diffuse readily into the extramitochondrial cytosol,

Glucose

Glucose 6-phosphate

Palmitate

Glucose

Palmitate

Glucose 6-phosphate

Malonyl Coa Palmitate

Figure 21-4. The provision of acetyl-CoA and NADPH for lipogenesis. (PPP, pentose phosphate pathway; T, tricarboxylate transporter; K, a-ketoglutarate transporter; P, pyruvate transporter.)

Figure 21-4. The provision of acetyl-CoA and NADPH for lipogenesis. (PPP, pentose phosphate pathway; T, tricarboxylate transporter; K, a-ketoglutarate transporter; P, pyruvate transporter.)

the principal site of fatty acid synthesis. Citrate, formed after condensation of acetyl-CoA with oxaloacetate in the citric acid cycle within mitochondria, is translocated into the extramitochondrial compartment via the tricarboxylate transporter, where in the presence of CoA and ATP it undergoes cleavage to acetyl-CoA and oxaloacetate catalyzed by ATP-citrate lyase, which increases in activity in the well-fed state. The acetyl-CoA is then available for malonyl-CoA formation and synthesis to palmitate (Figure 21-4). The resulting oxaloacetate can form malate via NADH-linked malate dehydrogenase, followed by the generation of NADPH via the malic enzyme. The NADPH becomes available for lipogenesis, and the pyruvate can be used to regenerate acetyl-CoA after transport into the mitochondrion. This pathway is a means of transferring reducing equivalents from extramitochondrial NADH to NADP. Alternatively, malate itself can be transported into the mitochondrion, where it is able to re-form oxaloacetate. Note that the citrate (tricarboxylate) transporter in the mitochondrial membrane requires malate to exchange with citrate (see Figure 12-10). There is little ATP-citrate lyase or malic enzyme in ruminants, probably because in these species acetate (derived from the rumen and activated to acetyl CoA extramitochondri-ally) is the main source of acetyl-CoA.

Elongation of Fatty Acid Chains Occurs in the Endoplasmic Reticulum

This pathway (the "microsomal system") elongates saturated and unsaturated fatty acyl-CoAs (from C10 upward) by two carbons, using malonyl-CoA as acetyl donor and NADPH as reductant, and is catalyzed by the microsomal fatty acid elongase system of enzymes (Figure 21-5). Elongation of stearyl-CoA in brain increases rapidly during myelination in order to provide C22 and C24 fatty acids for sphingolipids.

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