Oxaloacetate fatty acid synthesis

FIGURE 20.23 Export of citrate from mitochondria and cytosolic breakdown produces oxaloacetate and acetyl-CoA. Oxaloacetate is recycled to malate or pyruvate, which re-enters the mitochondria. This cycle provides acetyl-CoA for fatty acid synthesis in the cytosol. 

The acetyl-CoA derived from amino acid degradation is normally insufficient for fatty acid biosynthesis, and the acetyl-CoA produced by pyruvate dehydrogenase and by fatty acid oxidation cannot cross the mitochondrial membrane to participate directly in fatty acid synthesis. Instead, acetyl-CoA is linked with oxaloacetate to form citrate, which is transported from the mitochondrial matrix to the cytosol (Figure 25.1). Here it can be converted back into acetyl-CoA and oxaloacetate by ATP-citrate lyase. In this manner, mitochondrial acetyl-CoA becomes the substrate for cytosolic fatty acid synthesis. (Oxaloacetate returns to the mitochondria in the form of either pyruvate or malate, which is then reconverted to acetyl-CoA and oxaloacetate, respectively.)... 

Glycolysis, the pentose phosphate pathway, and fatty acid synthesis are all found in the cytosol. In gluconeo-genesis, substrates such as lactate and pyruvate, which are formed in the cytosol, enter the mitochondrion to yield oxaloacetate before formation of glucose. 

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. 

Calculating energy costs for the synthesis of a CK, fatty acid from acetyl-CoA is not as simple as you might first think. The major complication is that acetyl-CoA is made in the mitochondria, but fatty acid synthesis occurs in the cytosol—acetyl-CoA can t cross the mitochondrial membrane. Acetyl-CoA gets out of the mitochondria disguised as citrate. The acetyl-CoA is condensed with oxaloacetate to give citrate, and the citrate leaves the mitochondria. In the cytosol, the citrate is cleaved by an ATP-dependent citrate lyase into acetyl-CoA and oxaloacetate ... 

In common with cholesterol synthesis described in the next section, fatty acids are derived from glucose-derived acetyl-CoA. In the fed state when glucose is plentiful and more than sufficient acetyl-CoA is available to supply the TCA cycle, carbon atoms are transported out of the mitochondrion as citrate (Figure 6.8). Once in the cytosol, citrate lyase forms acetyl-CoA and oxaloacetate (OAA) from the citrate. The OAA cannot re-enter the mitochondrion but is converted into malate by cytosolic malate dehydrogenase (cMDH) and then back into OAA by mitochondrial MDH (mMDH) Acetyl-CoA remains in the cytosol and is available for fatty acid synthesis. 

The citrate shuttle transports acetyl CoA groups from the mitochondria to the cytoplasm for fatty acid synthesis. Acetyl CoA combines with oxaloacetate in the mitochondria to form citrate, but rather than continuing in the citric add cycle, citrate is transported into the cytoplasm. Factors that indirectly promote this process indude insuKn and high-energy status. 

Figure 11.3 Mechanism of transfer of acetyl-CoA out of the mitochondrion. In the mitochondrion, acetyl-CoA reacts with oxaloacetate to form citrate, which is transported across the mitochondrial inner membrane. In the cytosol, citrate is split to re-form citrate and oxaloacetate, catalysed by citrate lyase. It has been shown that inhibition of citrate lyase inhibits fatty acid synthesis.

Alternate fates of pyruvate Compounds other than lactate to which pyruvate can be converted ALTERNATE FATES OF PYRUVATE (p. 103) Pyruvate can be oxidatively decarboxylated by pyruvate dehydrogenase, producing acetyl CoA—a major fuel for the tricarboxylic acid cycle (TCA cycle) and the building block for fatty acid synthesis. Pyruvate can be carboxylated to oxaloacetate (a TCA cycle intermediate) by pyruvate carboxylase. Pyruvate can be reduced by microorganisms to ethanol by pyruvate decarboxylase. 

The biological effect of (-)-HCA stems from the inhibition of extramitochondrial cleavage of citrate to oxaloacetate and acetyl-CoA catalysed by ATPicitrate lyase. This limits the availability of acetyl-CoA units required for fatty acid synthesis and lipogenesis (Sullivan, 1984). The inhibition of ATP cit-rate lyase by (-)-HCA leads to less dietary carbohydrate utilization for the synthesis of fatty acids, resulting in more glycogen storage in the liver and muscles. Many in vitro... 

Citrate 2-Oxoglutarate Succinyl-CoA Malate Oxaloacetate Fatty acid biosynthesis Synthesis of glutamate Heme biosynthesis Gluconeogenesis Synthesis of aspartate... 

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 animals and fungi there is a similar dichotomy. NADPH can be generated by cytosolic malic enzyme which catalyses the reaction malate + NADP+ — pyruvate + COg + NADPH. Cytosolic malate derives from the following successive reactions the pyruvate/ citrate shuttle on the mitochondrial inner membrane takes pyruvate to the mitochondrion in exchange for citrate cytosolic ATP citrate lyase catalyses ATP + citrate + CoA-SH —> acetylCoA (CH3CO-S-C0A) + oxaloacetate and cytosolic malate dehydrogenase, which catalyses NADH + oxaloacetate NAD+ + malate. This scheme provides both acetylCoA and NADPH for subsequent long chain fatty acid synthesis (see section on Fatty acid synthesis ). 

Fatty acids are predominantly formed in the liver and adipose tissne, as well as the mammary glands during lactation. Fatty acid synthesis occurs in the cytosol (fatty acid oxidation occurs in the mitochondria compartmentalisation of the two pathways allows for distinct regulation of each). Oxidation or synthesis of fats utilises an activated two-carbon intermediate, acetyl-CoA, but the acetyl-CoA in fat synthesis exists temporarily bound to the enzyme complex as malonyl-CoA. Acetyl-CoA is mostly produced from pyruvate (pyruvate dehydrogenase) in the mitochondria it is condensed with oxaloacetate to form citrate, which is then transported into the cytosol and broken down to yield acetyl-CoA and oxaloacetate (ATP citrate lyase). 

Pyruvate Carboxylase Pyruvate carboxylase catalyzes the car-boxylation of pyruvate to oxaloacetate - both the first committed step of gluconeogenesis from pyruvate and also an important anaplerotic reaction, permitting repletion of tricarboxylic acid cycle intermediates and hence fatty acid synthesis. The mammalian enzyme is activated aUosterically by acetyl CoA, which accumulates when there is a need for increased activity of pyruvate carboxylase to synthesize oxaloacetate to permit increased citric acid cycle activity or for gluconeogenesis (Attwood, 1995 Jitrapakdee and Wallace, 1999). 

Pyruvate carboxylase is also important in lipogenesis. Citrate is transported out of mitochondria and cleaved in the cytosol to provide acetyl CoA for fatty acid synthesis the resultant oxaloacetate is reduced to malate, which undergoes oxidative decarboxylation to pyruvate, a reaction that provides at least half of the NADPH required for fatty acid synthesis. Pyruvate reenters the mitochondria and is carboxylated to oxaloacetate to maintain the process. 

Oxaloacetate formed in the transfer of acetyl groups to the cytosol must now be returned to the mitochondria The inner mitochondrial membrane is impermeable to oxaloacetate. Hence, a series of bypass reactions are needed. Most important, these reactions generate much of the NADPH needed for fatty acid synthesis. First, oxaloacetate is reduced to malate by NADH. This reaction is catalyzed by a malate dehydrogenase in the cytosol. 

In known metabolic states and disorders, the nature of metabolites excreted at abnormal levels has been identified by GC-MS. Examples of this are adipic and suberic acids found in urine from ketotic patients [347], 2-hydroxybutyric acid from patients with lactic acidosis [348], and methylcitric acid (2-hydroxybutan-l,2,3-tricarboxylic acid) [349] in a case of propionic acidemia [350,351]. In the latter instance, the methylcitric acid is thought to be due to the condensation of accumulated propionyl CoA with oxaloacetate [349]. Increased amounts of odd-numbered fatty acids present in the tissues of these patients due to the involvement of the propionyl CoA in fatty acid synthesis, have also been characterised [278]. A deficiency in a-methylacetoacetyl CoA thiolase enzyme in the isoleucine pathway prevents the conversion of a-methylacetoacetyl CoA to propionyl CoA and acetyl CoA [352,353]. The resultant urinary excretion of large amounts of 2-hydroxy-3-methylbutanoic acid (a-methyl-/3-hydroxybutyric acid) and an excess of a-methylacetoacetate and often tiglyl glycine are readily detected and identified by GC-MS. 

The reactions of fatty acid synthesis all take place in the cytosol, but acetyl-CoA is made in the mitochondria and can t cross the inner membrane. The Pyruvate-Malate Cycle (Citrate-Pyruvate Cycle) is used to take acetyl- groups to the cytosol while simultaneously providing a source of NADPH from NADH, and thus, coupling fatty acid synthesis to Glycolysis (Fig. 10.7). Note that the acetyl-CoA is first joined to oxaloacetate to make citrate which is readily transported out of the mitochondria using a co-transporter. The citrate is then cleaved to acetyl-CoA and oxaloacetate, a process requiring ATP to make it favourable (recall the condensation was spontaneous). Acetyl-CoA for fatty acid synthesis is now available in the cytosol, but oxaloacetate must be regenerated for the mitosol. 

Carboxylation. Gluconeogenesis begins with the carboxylation of pyruvate to yield oxaloacetate. As in the third step of fatty acid synthesis (Figure 29.8), the reaction requires ATP and the coenzyme biotin, acting as a carrier of CO2. 

Because acetyl CoA cannot directly cross the mitochondrial membrane and enter the cytosol to be used for the process of fatty acid synthesis, acetyl CoA and oxaloacetate condense to form citrate, which can cross the mitochondrial membrane. 

C. Fatty acid synthesis is maximal in the fed state when insulin is elevated. Glucose is converted to citrate, which is cleaved to oxaloacetate and acetyl CoA. The regulatory enzyme... 

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... 

Pyruvate has several metabolic fates. It can be reduced to lactate, converted to oxaloacetate in a reaction important in gluconeogenesis (Chapter 15) and in an anaplerotic reaction of the TC A cycle (see below), transminated to alanine (Chapter 17), or converted to acetyl-CoA and CO2. Acetyl-CoA is utilized in fatty acid synthesis, cholesterol (and steroid) synthesis, acetylcholine synthesis, and the TCA cycle (Figure 13-6). 

Thus, citrate not only modulates the rate of fatty acid synthesis but also provides carbon atoms for the synthesis. The oxaloacetate formed from pyruvate may eventually be converted (via malate) to glucose by the gluconeogenic pathway. The glucose oxidized via the pentose phosphate pathway augments fatty acid synthesis by providing NADPH. Pyruvate generated from oxaloacetate can enter mitochondria and be converted to oxaloacetate, which is required for the formation of citrate. 

Tie reactions constitute a metabolic motif that we will see again in fatty acid synthesis and degradation as well as in the degradation of some amino acids. A methylene group (CH2) is converted into a carbonyl group (C=0) in three steps an oxidation, a hydration, and a second oxidation reaction, Oxaloacetate is thereby regenerated for another round of the cycle, and more energy is extracted in the form of FADH and NADH. 

The accumulation of the precursors for fatty acid synthesis is a wonderful example of the coordinated use of multiple pathways. The citric acid cycle, transport of oxaloacetate from the mitochondria, and pentose phosphate pathway provide the carbon atoms and reducing power, whereas glycolysis and oxidative phosphorylation provide the ATP to meet the needs for fatty acid synthesis (Figure 22.28). 

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