ATP-dependent citrate lyase

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 ... 

The tricarboxylic acid cycle not only takes up acetyl CoA from fatty acid degradation, but also supplies the material for the biosynthesis of fatty acids and isoprenoids. Acetyl CoA, which is formed in the matrix space of mitochondria by pyruvate dehydrogenase (see p. 134), is not capable of passing through the inner mitochondrial membrane. The acetyl residue is therefore condensed with oxaloacetate by mitochondrial citrate synthase to form citrate. This then leaves the mitochondria by antiport with malate (right see p. 212). In the cytoplasm, it is cleaved again by ATP-dependent citrate lyase [4] into acetyl-CoA and oxaloacetate. The oxaloacetate formed is reduced by a cytoplasmic malate dehydrogenase to malate [2], which then returns to the mitochondrion via the antiport already mentioned. Alternatively, the malate can be oxidized by malic enzyme" [5], with decarboxylation, to pyruvate. The NADPH+H formed in this process is also used for fatty acid biosynthesis. 

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. 

A second ATP-dependent citrate lyase (ACL) activity is responsible for the formation of acetyl-CoA 30 and oxaloacetate 35 from citrate 39 with concomitant hydrolysis of ATP to ADP and phosphate (Equation (22)). ° It is proposed to play a vital role in maintaining acetyl-CoA and oxaloacetate levels in most mammals, whereas in some bacteria it is an essential enzyme of the reductive tricarboxylic acid cycle (RTCA). 

Citrate that is not oxidized by isocitrate dehydrogenase can be transported from the mitochondrial matrix into the cytoplasm. In the cytoplasm of adipocytes and hepatocytes, oxaloacetate and acetyl-CoA are formed from citrate, not by the reversal of the citrate synthase-catalyzed reaction, bnt by ATP-dependent citrate lyase. As the name indicates, the free energy of ATP hydrolysis drives this reaction in the degradative direction. 

A temporary deficiency of oxaloacetate in the matrix reduces the rate of disposal of acetyl-CoA, but cytosolic oxaloacetate produced via ATP-dependent citrate lyase returns to the matrix, thus overcoming the deficiency. Oxaloacetate itself does not cross the inner mitochondrial membrane it is converted to compounds for which there are specific transporters. 

There it is cleaved by ATP-citrate lyase. To ensure that the reaction goes to completion, cleavage is coupled to the hydrolysis of ATP to ADP and inorganic phosphate (Eq. 13-39). The value of G given here is extremely dependent upon the concentration of Mg2+ as a result of strong chelation of Mg2+ by citrate.220 The reaction sequence is complex but can be understood in terms of an initial ATP-dependent synthesis of... 

Three modifications of the conventional oxidative citric acid cycle are needed, which substitute irreversible enzyme steps. Succinate dehydrogenase is replaced by fumarate reductase, 2-oxoglutarate dehydrogenase by ferredoxin-dependent 2-oxoglutarate oxidoreductase (2-oxoglutarate synthase), and citrate synthase by ATP-citrate lyase [3, 16] it should be noted that the carboxylases of the cycle catalyze the reductive carboxylation reactions. There are variants of the ATP-driven cleavage of citrate as well as of isocitrate formation [7]. The reductive citric acid... 

Incubation of hepatocytes with glucagon also increases the phosphorylation of another lipogenic enzyme ATP-citrate lyase [127,128], The purified enzyme is also phosphorylated by cAMP-dependent protein kinase in vitro [128,129] resulting in a 2-fold increase in the Km for ATP [130], However, it is unclear what role this plays in the inhibition of lipogenesis. 

Glucagon decreases cholesterol synthesis in isolated hepatocytes [131,132] apparently because it reduces the fraction of hydroxymethylglutaryl-CoA reductase in the active form [131,132], This is due to an increase in reductase kinase activity [133], However, there is no evidence that cAMP-dependent protein kinase phos-phorylates either the reductase, reductase kinase or reductase kinase kinase [134], It has been proposed that the phosphorylation state of these enzymes is indirectly controlled through changes in the activity of protein phosphatase I [132,134], This phosphatase can dephosphorylate and activate the reductase [134,135] and its activity can be controlled by a heat stable inhibitor (inhibitor 1), the activity of which is increased by cAMP-dependent phosphorylation [136,137], Since the phosphorylated forms of acetyl-CoA carboxylase, ATP-citrate lyase, pyruvate kinase, phos-phorylase, phosphorylase kinase and glycogen synthase are also substrates for protein phosphatase I [135], this mechanism could also contribute to their phosphorylation by glucagon. 

Glucagon affects hepatic lipid metabolism. A major effect is inhibition of fatty acid synthesis, which is mainly due to the phosphorylation and inhibition of acetyl-GoA carboxylase by cAMP-dependent protein kinase. ATP-citrate lyase is also phosphorylated, but it is unclear that this is involved in the inhibition of lipogene-sis. Glucagon also inhibits cholesterol synthesis apparently due to a decrease in the activity of hydroxymethylglutaryl-CoA reductase. This is thought to result from a decrease in the activity of protein phosphatase I due to the increased phosphorylation and activation of a heat stable inhibitor by cAMP-dependent protein kinase. This mechanism could also contribute to the effects of glucagon on other hepatic enzymes. 

Scheme 5 Malonate decarboxylase and citrate lyase both contain a bo/o-ACP (MdcC and CitD, respectively) that has a 2 -(5"-phosphoribosyl)-3 -dephospho-CoA prosthetic group which is attached by a phosphodiester linkage to a conserved serine residue. The prosthetic group originates from the cofactor 2 -(5"-triphosphoribosyl)-3 -dephospho-CoA 24, which is biosynthesized from dephospho-CoA 11 and ATP by either MdcB or CitG. The posttranslational modification of theapo-ACP proteins is catalyzed by a complex of the enzyme MdcG with 24, or CitX, depending on the system.

Why is synthesis of cholesterol de novo dependent on the activity of ATP-citrate lyase ... 

Microorganisms lacking ATP citrate lyase do not accumulate lipid much above 10-15%, although the corollary, that organisms with ATP citrate lyase activity will accumulate lipid may not always hold because other enzymes, such as AMP deaminase, AMP-dependent isocitrate dehydrogenase or malic enzyme, may be lacking or may not be under sufficiently stringent control to allow extensive lipid accumulation. There is clearly much still to be learnt about lipid accumulation and its attendant biochemistry to explain all these variations at the fundamental level. 

Carbon for FAS could also be produced via ATP-citrate lyase. This activity, which will convert citrate to oxaloacetate plus acetyl-CoA, has been demonstrated in soybean extracts. But there is no current evidence for citrate transport into the plastid nor of localisation of this activity in the plctstid to support citrate cleaving enzyme as a source of carbon for FAS, at least for avocado mesoccirp plastids. Extracts of developing soybean also contain a NADP+-dependent medic enzyme.20 NAD+-dependent malic enzyme, which produces pyruvate ind Cctrbon dioxide from malate, is an enzyme specific to the mitochondrial matrix in higher pleints.21 The localisation of NADP+-malic enzyme in immature soybeans, eind the possibility of pyruvate production other than by pyruvate kinase, and the utilisation of this pyruvate in FAS, remain to be determined. 

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