Rate limiting step of fatty acid synthesis

Fatty Acid Synthesis

Fatty acid synthesis occurs in the cytosol and is regulated closely by the availability of acetyl-CoA, which forms the basic subunit of the developing fatty acid carbon chain.93 Acetyl-CoA is synthesized predominantly in mitochondria and is derived mainly from carbohydrate metabolism, with a small fraction coming from amino acids.6, 14, 15 Acetyl-CoA is condensed with oxaloacetate to form citrate, which is exported from the mitochondria and is then cleaved by the cytosolic ATP citrate lyase to produce oxaloacetate and acetyl-CoA. Conversion of acetyl-CoA to malonyl-CoA by the action of acetyl-CoA carboxylase is the first step in fatty acid synthesis. Acetyl-CoA carboxylase is the key enzyme in regulating fatty acid synthesis because it provides the necessary building blocks for elongation of the fatty acid carbon chain.115

Malonyl-CoA is used by a set of enzymatic activities contained within a single peptide chain that comprises the remarkable fatty acid synthase system.93 Malonyl-CoA binds to acyl carrier protein. Catalytic activity is contained within 2 distinct domains that catalyze sequential condensation, reduction, dehydrogenation, and reduction, which constitute the fatty acid synthetic cycle. Two nicotinamide dinucleotide phosphate molecules are required for each 2-carbon unit that is added to the growing fatty acid chain. After completion of the first cycle, the 4-carbon butyl group is transferred from acyl carrier protein to a peripheral thiol, thereby allowing it to accept the next malonyl-CoA group to restart the entire cycle. The cycle continues for an additional 6 or 7 rounds until a carbon-16 (palmitate) or carbon-18 (stearate) fatty acid is synthesized. Fatty acid-CoA is then released and used for other metabolic pathways.

Further elongation of the fatty acid chain can occur either in the mitochondrion or within the microsomal membrane.93 In the mitochondrion, the first step is mediated by enoyl-CoA reductase. Microsomal elongation uses malonyl-CoA to increase the size of fatty acyl-CoA in a process that involves 4 separate enzymatic reactions. The elongation ability of microsomes is tissue dependent and serves the needs of specific organs. The fatty acid chain elongates until an appropriate length has been achieved, and the fatty acid is then esterified with glycerol to form TG. These newly formed TG can be transported by lipoproteins to distal sites for storage and use. In situations of excess carbohydrates, PYR can be converted to acetyl-CoA by the mitochondrial pyruvate dehydrogenase complex to serve as fatty acid precursors, although lipogenesis from carbohydrates consumes about 25% of the energy contained in the carbohydrates.

4.3 Production of NADPH

Fatty acid synthesis utilizes two molecules of NADPH for each molecule of acetate incorporated into long-chain fatty acids. In liver, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (Fig. 1) probably furnish about half of the NADPH used in fatty acid synthesis, with the other half coming from malic enzyme. The activities of the two dehydrogenases of the pentose phosphate pathway and of malic enzyme correlate positively with the rate of fatty acid synthesis under a wide variety of conditions. However, the rate of production of NADPH does not regulate fatty acid synthesis. In liver, each of these enzymes is usually near equilibrium with respect to its substrates and products, and thus, changes in the activities of these enzymes do not alter the rate of production of NADPH. The rate of production of NADPH is thus a function of its utilization.

Carboxylation of acetyl-CoA to malonyl-CoA is the committed step of fatty acid synthesis

In the first stage of fatty acid biosynthesis, acetyl-CoA,mostly derived from carbohydrate metabolism, is converted to malonyl-CoA by the action of the enzyme acetyl-CoA carboxylase (Fig. 13.1). There are two forms of acetyl-CoA carboxylase (ACC1 and ACC2).ACC1 is located in the cytoplasm and committed to fatty acid synthesis, whereasACC2 is in mitochondria, where it regulates fatty acid oxidation. ACC2 inhibition results in reduced generation of malonyl-CoA, which in turn is an inhibitor of the carnitine-palmitoyl transferase 1 (CPT-1) allowing fatty acid uptake by the mitochondria. Through this inhibition, the fatty acid oxidation is reduced. ACC1 is a biotin-dependent enzyme with distinct enzymatic and carrier-protein function: its subunits serve as a biotin carboxylase, transcarboxylase, and biotin carboxyl carrier protein. The enzyme is synthesized in an inactive protomer form, with each protomer containing all of the previously described subunits, a molecule of biotin, and a regulatory allosteric site for the binding of citrate (a Krebs cycle metabolite) or palmitoyl-CoA (the end product of the fatty acid biosynthetic pathway). The reaction itself takes place in stages: first, there is the carboxylation of biotin, involving adenosine triphosphate (ATP), followed by the transfer of this carboxyl group to acetyl-CoA to produce the malonyl-CoA. At this stage, the free enzyme–biotin complex is released.

As described previously, this process only allows the building up of fatty acid molecules with even numbers of carbon atoms, which is seen in eukaryote cells. However, propionyl-CoA is a substrate for the synthesis of fatty acids with an odd number of carbon atoms, but this is not seen in humans. Typically, the odd-chain fatty acids seen in humans originate from milk fat consumption because the bacterial/fermentation process in ruminants allows for the production of these fatty acids.

10.5 Coordinate regulation of fatty acid and phospholipid synthesis with macromolecular biosynthesis

Fatty acid biosynthesis is coordinately regulated with phospholipid synthesis. Labeling the ACP moiety of the fatty acid intermediates by growth of a panD strain on medium containing tritiated β-alanine, a precursor of the 4′-phosphopantetheine ACP prosthetic group, shows that long-chain acyl-ACPs accumulate for a short period following the cessation of phospholipid synthesis. This accumulation does not continue indefinitely, however, and reaches a plateau after about 20 min following inhibition of phospholipid synthesis. Thus, de novo fatty acid synthesis ceases, probably by a feedback inhibition mechanism involving long-chain acyl-ACPs inhibiting early steps in the fatty acid biosynthesis pathway (Fig. 10). A significant finding in support of this idea is that overexpression of a thioesterase (which prevents the accumulation of acyl-ACP by cleavage of the thioester linkage and releases the acyl chain) allows continued fatty acid synthesis following cessation of phospholipid synthesis. This further suggests that acyl-ACP and not free fatty acids mediate the regulation. A reduction in total ACP is not responsible for the inhibition of fatty acid synthesis, since the free ACP pools of the glycerol-starved plsB mutants are not significantly depleted, and overproduction of ACP fails to relieve inhibition of fatty acid synthesis. A fadD mutant strain, which cannot produce acyl-CoA, overexpressing a thioesterase gave the same results as strains blocked elsewhere in β-oxidation or wild-type strains, thus ruling out a role for acyl-CoA.

There are several target enzymes for acyl-ACP feedback inhibition (Fig. 10). ACC is an obvious target and indeed long-chain acyl-ACP inhibits the activity of this enzyme. Because malonyl-CoA is required for both initiation and elongation of fatty acids, a blockade at this step is very effective. Acyl-ACP also inhibits FabH, which catalyzes the first step in the pathway. Inhibition of this enzyme would halt initiation of new acyl chains, but would allow the elongation of existing fatty acid intermediates. Inhibition of FabH by physiologically relevant concentrations of long-chain acyl-ACPs has been demonstrated in vitro. Finally, enoyl-ACP reductase (FabI) is an acyl-ACP target and is important because the activity of this enzyme is a determining factor in completing rounds of fatty acid elongation. Acyl-ACP acts as a product inhibitor of FabI. The relative contributions of each of these regulatory points to the control of fatty acid synthesis in vivo is not clear, but it is likely that they all contribute to the cessation of acetate incorporation following the accumulation of acyl-ACP.

Fatty acid synthesis in E. coli is also regulated by an unusual nucleotide, guanosine 5′-diphosphate-3′-diphosphate (ppGpp) [23]. Wild-type strains of E. coli undergo the so-called ‘stringent response’ following starvation for a required amino acid, an effect also mediated by increased intracellular ppGpp. Increased levels of ppGpp cause a strong inhibition of stable RNA synthesis and inhibition of protein and phospholipid synthesis. Mutant strains (relA) do not undergo the stringent response following amino acid starvation, due to the lack of ppGpp synthase I, a ribosomal protein that produces pppGpp in response to uncharged tRNA. The interaction of ppGpp with RNA polymerase mediates the inhibitory effects on stable RNA synthesis. ppGpp directly inhibits phospholipid biosynthesis by inhibition of the PlsB and causes an accumulation of long-chain acyl-ACPs, which in turn lead to the inhibition of fatty acid biosynthesis. Overexpression of the acyltransferase relieves the inhibition on both fatty acid and phospholipid synthesis. These events are regulated by ppGpp formation by ppGpp synthase II, or SpoT. Intriguingly, a direct interaction occurs between ACP and SpoT that is likely to play a key role in regulating ppGpp production in response to the status of fatty acid biosynthesis. However, there are many biochemical details that need to be worked out to establish the mechanism for this regulation.

7.27.5.1.8.1 Fatty acid biosynthesis

Fatty acid biosynthesis is important for cell growth, differentiation, and homoeostasis. Unlike in animals and fungi, where a single multifunctional enzyme known as type I fatty acid synthase catalyzes all, the steps in fatty acid biosynthesis, bacteria, plants, and Plasmodium spp. use a type II fatty acid biosynthetic pathway (Figure 8) in which each step is catalyzed by separate enzymes.389

Table 9 lists some of the compounds identified thus far that inhibit various enzymes involved during fatty acid biosynthesis. It is interesting to note that one of these compounds, triclosan (94), a known inhibitor of FabI and a widely used antibacterial agent,390 inhibited P. falciparum growth in vitro with an IC50 value of 0.7 μM. When tested in vivo in mice infected with P. berghei at a 3.0 mg kg−1 dose subcutaneously, this compound inhibited parasitemia by 75% within 24 h of drug administration.391 Compounds Genz-8575 (97) and Genz-10850 (98) were identified by high-throughput screening of a library of compounds against enoyl acyl carrier protein reductase of Mycobacterium tuberculosis (inhA). In addition to inhA, they also inhibited the corresponding Plasmodium enzyme (PfENR) with IC50 values of 32 μM and 18 μM, respectively, and were active against P. falciparum growth in vitro (Table 9).392 Structural optimization on various compounds identified already, and future design based on the active site details of enzymes involved in fatty acid synthesis are expected to give better candidates for medicinal use.

Table 9. Compounds and their targets involved in the fatty acid synthesis in malaria parasites

CompoundProposed target(s)P. falciparum growth inhibition (IC50)ReferenceACC144±22 μMWaller et al.393Fab B/F/H49±8 μMWaller et al.393Fab B/F/H8±0.2 μMWaller et al.393FabZ7.4 μMSharma et al.394FabI0.7 μMSurolia et al.391FabI1.8–6.0 μMPerozzo et al.395FabI5.9–19.5 μMPerozzo et al.395FabI10–16 μMKuo et al.392FabI14–32 μMKuo et al.392

Metabolic Roles/Functions

CoA and its derivatives function in a wide variety of metabolic pathways including the tricarboxylic cycle, fatty acid oxidation, ketogenesis, fatty acid biosynthesis, sterol synthesis, complex lipid synthesis, amino acid metabolism, and porphyrin synthesis. (Since most of these are covered elsewhere in this encyclopedia, they are not dealt with in this entry.) As the fatty acid synthase system is unique, in that it makes use of only a fragment of the CoA molecule, 4′-PP, a brief discussion of FAS is warranted.