Source of acetyl-CoA

The synthesis of fatty acids and sterols in the liver cytosol depends upon a common pool of acetyl-CoA. This was demonstrated by Decker and Barth in a series of experiments utilizing perfused rat liver [10]. Lipid synthesis was measured by incorporation of tritium from [ H]H20. They used (- )-hydroxycitrate to inhibit ATP-dependent citrate lyase and measured radioisotope incorporation into fatty acids and sterols as a function of the concentration of this inhibitor. A parallel decrease in incorporation into these two products was found as the concentration of (- )-hydroxycitrate in the perfusate was increased. Contrastingly, if radioisotopic acetate was used as the substrate in the perfusing medium, this inhibitor had relatively little effect on the rate of sterologenesis, a result that would be expected if the natural source of acetate was from the action of the cytoplasmic citrate lyase. Their experiments also demonstrated that the ratio of fatty acid synthesis to sterol synthesis in the liver of fed rats is about 10 1. 

Acetoacetate is another vehicle for transporting acetyl groups into the cytoplasm. This molecule, one of the end products of ketone body synthesis, is free to diffuse from the mitochondrion. When in the cytoplasm it can be activated to acetoacetyl-CoA by an ATP-dependent acetoacetyl-CoA synthetase. Edmonds group has shown that the activity of this enzyme parallels the rate of cholesterologenesis in the Uvers of animals given a variety of dietary regimes [11]. Their data also indicate that this pathway furnishes as much as 10% of the carbon required for cholesterol biosynthesis. 

Several other pathways have been postulated for the transport of acetyl units from the mitochondrion to the cytoplasm. One entails carnitine as a transporter, as it is for fatty acids, the other invoked free acetate. It is unlikely that either of these is significant [12]. 

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Pyruvate produced by glycolysis is a significant source of acetyl-CoA for the TCA cycle. Because, in eukaryotic ceils, glycolysis occurs in the cytoplasm, whereas the TCA cycle reactions and ail subsequent steps of aerobic metabolism take place in the mitochondria, pyruvate must first enter the mitochondria to enter the TCA cycle. The oxidative decarboxylation of pyruvate to acetyl-CoA,... 

There are three principal sources of acetyl-CoA (Figure 25.1) ... 

Citrate is isomerized to isocitrate by the enzyme aconitase (aconitate hydratase) the reaction occurs in two steps dehydration to r-aconitate, some of which remains bound to the enzyme and rehydration to isocitrate. Although citrate is a symmetric molecule, aconitase reacts with citrate asymmetrically, so that the two carbon atoms that are lost in subsequent reactions of the cycle are not those that were added from acetyl-CoA. This asymmetric behavior is due to channeling— transfer of the product of citrate synthase directly onto the active site of aconitase without entering free solution. This provides integration of citric acid cycle activity and the provision of citrate in the cytosol as a source of acetyl-CoA for fatty acid synthesis. The poison fluo-roacetate is toxic because fluoroacetyl-CoA condenses with oxaloacetate to form fluorocitrate, which inhibits aconitase, causing citrate to accumulate. 

Supplies P, for oxidative phosphorylation Shuttles reducing equivalents (as malate) from matrix to cytosol Completes shuttling begun by malate-a-ketoglutarate shuttle Provides cytosolic citrate as source of acetyl-CoA for lipid synthesis... 

The biosynthesis of fatty acids such as palmitate thus requires acetyl-CoA and the input of chemical energy7 in two forms the group transfer potential of ATP and the reducing power of NADPH. The ATP is required to attach C02 to acetyl-CoA to make malonyl-CoA the NADPH is required to reduce the double bonds. We return to the sources of acetyl-CoA and NADPH soon, but first let s consider the structure of the remarkable enzyme complex that catalyzes the synthesis of fatty acids. 

The condensation of acetyl CoA and oxaloacetate to form citrate is catalyzed by citrate synthase (Figure 9.5). This aldol condensation has an equilibrium far in the direction of citrate synthesis. Citrate synthase is allosterically activated by Ca2+ and ADP, and inhibited by ATP, NADH, succinyl CoA, and fatty acyl CoA derivatives (see Figure 9.9). However, the primary mode of regulation is also deter mined by the availability of its substrates, acetyl CoA and oxaloac etate. [Note Citrate, in addition to being an intermediate in the TCA cycle, provides a source of acetyl CoA for the cytosolic synthesis of... 

It is an acyl-CoA of the type mentioned in Section 1 and can also be formed from acetate, ATP, and coenzyme A. Although the human diet contains some acetic acid, the two major sources of acetyl-CoA in our bodies are the oxidative decarboxylation of pyruvate (Eq. 10-6) and the breakdown of fatty acid chains. Let us consider the latter process before examining the further metabolism of acetyl-CoA. 

The acetyl-CoA produced in the cytosol from citrate breakdown is used in biosynthetic reactions, including the synthesis of lipids, some amino acids, cofactors, and pigments. This is the only source of acetyl-CoA in the cytosol because the acetyl-CoA produced in the mitochondrion cannot diffuse across the mitochondrial membrane. The NADPH produced indirectly in the cytosol from the citrate is also of great importance in biosynthetic reactions. 

I- oxaloacetate + ADP + Pj. This reaction takes place in the cytoplasm and is a source of acetyl-CoA for fatty acid biosynthesis. 

One source of acetyl-CoA molecules for the citric acid cycle is via oxidation of pyruvate in a reaction catalyzed by the pyruvate dehydrogenase complex. The process of converting pyruvate to acetyl-CoA is an oxidative decarboxylation. In the overall reaction (below), the carboxyl group of pyruvate is lost as C02, while the remaining two carbons form the acetyl moiety of acetyl-CoA. 

One source of acetyl-CoA molecules for the citric acid cycle is oxidative decarboxylation of pyruvate, catalyzed by the pyruvate dehydrogenase complex. The complex is composed of three enzymes... 

The conversion of pyruvate to fatty acids requires a source of acetyl CoA in the cytosol. Pyruvate can only be converted to acetyl CoA in mitochondria, so it enters mitochondria and forms acetyl CoA through the pyruvate dehydrogenase (PDH) reaction. This enzyme is dephosphorylated and most active when its supply of substrates and adenosine diphosphate (ADP) is high, its products are used, and insulin is present (Fig. 36.3). 

An inherited pyruvate dehydrogenase deficiency, a thiamine deficiency, or hypoxia deprives the brain of a source of acetyl CoA for acetylcholine synthesis, as well as a source of acetyl CoA for ATP generation from the TCA cycle. 

If the mitochondrion is a principal site of the PDC, hence the formation of acetyl-CoA, then the plant cell must employ a transport system which can export this highly reactive substrate from the matrix of the mitochondrion to the appropriate site where it is utilized for fatty acid biosynthesis. However, this problem may not be serious, since in seeds that contain high levels of lipid at maturity the proplastid appears to be the principal site of the conversion of pyruvate to acetyl-CoA by the PDC and of its efficient utilization for the biosynthesis of palmitoyl-and/or stearoyl-ACP. In the chloroplast, evidence for the PDC is not clear, although indirect evidence does suggest PDC activity (Murphy and Leech, 1978) in these organelles. Since acetate moves freely across the chloroplast membrane and since acetyl-CoA synthetase occurs in the chloroplast stroma (Jacobson and Stumpf, 1972), it is possible that the primary source of acetyl-CoA derives from its synthesis at a site other than the chloroplast, its transport into the chloroplast as the undissociated acid and/or free anion, and the conversion of acetate back to acetyl-CoA by the stroma acetyl-CoA synthetase. Further work is necessary to clarify this important point. It is difficult to explain the function of acetyl-... 

Evidence has been presented that citrate is the source of acetyl-CoA in the soybean cotyledon. Nelson and Rinne (1975, 1977a,b) have described the occurrence of citrate lyase in the cytosol of developing soybean seeds. Furthermore, [1,5-1 ] citrate was an effective donor of [ KI ]acetyl-CoA for the synthesis of fatty acids in these extracts. However, Weaire and Kekwick (1975) did not find [l,5- K ]citrate to be an acetyl donor for fatty acid synthesis in avocado extracts. In addition, Yamada and Nakamura (1975) showed with isolated spinach chloroplasts that, whereas pyruvate was effectively incorporated into fatty acid, citrate, malate, and oxeiloacetate were far less active. Apparently different tissues may supply acetyl-CoA from different precursors, i.e., pyruvate, citrate, and acetate. 

In the ruminant animal, acetate is absorbed directly from the gut and is changed to acetyl-CoA, in the presence of acetyl-CoA synthetase (see p. 206), in the cell cytoplasm. This is the major source of acetyl-CoA in ruminants, in which ATP-citrate lyase activity is greatly reduced and passage of mitochondrial acetyl-CoA to the cytoplasm is limited. 

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