Liver acetyl-CoA

Takai, T., Yokoyama, C., Wada, K., and Tanabe, T. (1988). Primary structure of chicken liver acetyl-CoA carboxylase deduced from cDNA sequence./. Biol. Chem., 263, 2651-2657. 

Figure 19.12 Regulation of liver acetyl-CoA carboxylase and cholesterol biosynthesis by phosphorylation. ACC indicates acetyl-CoA carboxylase. Bold arrow indicates activation of kinase kinase by fatty acyl-CoA. (Reproduced by permission from Hardie DG, Carling D, Sim ATR. The AMP-activated protein kinase a multi-substrate regulator of lipid metabolism. Trends Biochem Sci 14 20-23, 1989.)...

Davies, S.P., Carling, D., Munday, M.R., and Hardie, D.G., 1992, Diurnal rhythm of phosphorylation of rat liver acetyl-CoA carboxylase by the AMP-activated protein kinase, demonstrated using freeze-clamping. Effects of high fat diets. Eur. J. Biochem. 203 615-623. 

In tissues such as skeletal and heart muscle, acetyl CoA enters the TCA cycle and is oxidized to C02 and H20. In the liver, acetyl CoA is converted to ketone bodies. 

Enzymatic regulation of liver acetyl-CoA metabolism in relation to ketogenesis, O. Wieland, L. Weiss, and I. Eger-Neufeldt, 1964, Adv. Enzyme Reg., 8599. 

Not all acetyl CoA generated from P-oxidation enters the TCA cycle. In the liver, acetyl CoA generated from p-oxidation of fatty acids can also be converted to the... 

Fig. 23.1. Overview of mitochondrial long-chain fatty acid metabolism. (1) Fatty acid binding proteins (FaBP) transport fatty acids across the plasma membrane and bind them in the cytosol. (2) Fatty acyl CoA synthetase activates fatty acids to fatly acyl CoAs. (3) Carnitine transports the activated fatty acyl group into mitochondria. (4) p-oxidation generates NADH, FAD(2H), and acetyl CoA (5) In the liver, acetyl CoA is converted to ketone bodies...

Fig. 4.4). Acetyl-CoA carboxylase exists as an inactive dimer x 500 kDa) and a polymeric active form ( 20 X 500 kDa). The allosteric effector palmitoyl-CoA competes with citrate and causes depolymerisation of acetyl-CoA carboxylase and inhibits its activity. Phosphorylation of acetyl-CoA carboxylase also causes its inactivation. The primary structure of domestic fowl liver acetyl-CoA carboxylase has been deduced from its cDNA sequence (Takai et ai, 1988). 

Acetyl-CoA carboxylase is also inhibited by long-chain fatty acyl-CoA, and such inhibition is accompanied by enzyme depolymerization (91, 92, 95). Binding of 1 mole of palmityl-CoA per mole of rat liver acetyl-CoA carboxylase inhibits the enzyme (95). The Ti for palmityl-CoA, about 5 nM, is far lower than the critical micellar concentration of the thioester this indicates that the inhibition may be physiologically significant. If the allosteric control mechanisms of citrate promoted "substrate activation or fatty acyl-CoA mediated "feed back inhibition of fatty acid synthesis function at all under in vivo conditions, they must function as a dual mechanism (90). 

In 1973 when Carlson and Kim reported that acetyl-CoA carboxylase was regulated by a covalent modification mechanism involving interconversion between the phosphorylated (inactive) and the dephos-phorylated form (active) 19), there was some suggestive evidence for such a mechanism in the literature. For example, several investigators had observed that cyclic AMP and dibutyryl cyclic AMP inhibit the incorporation of [ Clacetate or [ CJglucose into fatty acid (5,12, 44, 45). Also treatment of rat or chicken liver and rat epididymal fat tissues in vitro with cyclic nucleotides results in the inactivation of acetyl-CoA carboxylase 4, 69). In addition to these observations, Greenspan and Lowenstein reported that crude enzsrme preparations of rat liver acetyl-CoA carboxylase were inactivated in the presence of ATP and Mg + and the inactivation was reversed by incubation with Mg + 34). However, ATP- and Mg -mediated inactivation was often explained in terms of a hypothetical, unstable E Biotin CO2 species 25, 39, 64). Thus, considerable controversy persisted for many years as to the occurrence of the covalent modification mechanism until several investigators published supportive evidence for this mechanism in various tissues 3, 16, 19, 41, 42, 66, 68, 105, 129). 

Fig. 4. Phosphorylation profiles of rat liver acetyl-CoA carboxylase on SDS-acrylamide gels after various times of inactivation. The partially purified enzyme preparation was incubated with [ P]ATP (specific activity 84 /uCi/ mol) for var3ring periods. Subsequently, aliquots of P-labeled enzyme were purified further by DEAE-cellulose chromatography, precipitated with antibody made against the enzyme subunit, and subjected to electrophoresis on SDS-polyacrylamide gels. (A) O-O, 5-minute inactivation A----A, 10-minute inactivation. (B) -- , 20-minute inactivation --- ,...

However, cyclic AMP and its derivative, dibutyryl cyclic AMP, do inactivate the carboxylase under in vivo conditions. Therefore, the action of cyclic AMP is apparently not at the step of the activation of the carboxylase kinase, but at some other step leading to such activation. It is not entirely clear why the carboxylases from tissues other than mammary glands are not responsive to the cyclic AMP-dependent protein kinase. However, this lack of a cyclic AMP-dependent protein kinase effect could be related to experimental conditions since the suitability of the carboxylase as a substrate for the kinase is drastically aflFected hy cellular metabolites (see Section V). Allred and Harris (3) reported that the cyclic AMP-dependent phosphorylation site or entity dissociates itself from the carboxylase as the enzyme is purified. Thus, the purified preparation does not contain the site whose phosphorylation is stimulated by cyclic AMP. Two recently purified protein kinases active toward the liver acetyl-CoA carboxylase are cyclic AMP independent (70, 105). The properties of these kinases will be discussed later. 

Partial purification of cyclic AMP-independent protein kinase which phosphorylates and inactivates rat liver acetyl-CoA carboxylase has been reported recently (105) the molecular weight of the kinase is 160,000 (105). Four to five moles of phosphate were incorporated per mole of protomer, corresponding to about 2 mole of phosphate per mole of subunit. Similar values for phosphate incorporation were observed when the carboxylase was inactivated in the presence of endogenous kinase (71). 

It should be noted that during 3 hours of incubation with Pi, about 4800 cpm were incorporated although enzyme activity was not affected at all. This observation indicates that the phosphorylation which occurred in the absence of epinephrine has no functional relationship to enzyme activity. The phosphorylation which occurs in the presence of epinephrine, however, can inactivate carboxylase. Indeed, this phosphorylation of the carboxylase which is unrelated to enzyme activity may have misled some to conclude that the covalent phosphorylation of carboxylase has no physiological significance (25,39,99). Witters al. (129) have also provided evidence for the causal relationship between the phosphorylation and inactivation of rat liver acetyl-CoA carboxylase following glucagon treatment of isolated hepatocytes which had been prelabeled with Pi. 

The effects of citrate and isocitrate on the sedimentation behavior of the enzyme have been verified by direct electron microscopic examination. Electron microscopy of the avian liver acetyl-CoA carboxylase in the assay reaction mixture without tricarboxylic acid activator, in which the enzyme exists as the inactive 13 to 15 S species, reveals small protomeric forms having minimum and maximum dimensions of 70 A and 130 A, respectively (Fig. 7 4, reference [176]). Addition of either citrate or isocitrate, which activates the enzyme and leads to the rapidly sedimenting 47 to 50 S species, causes instantaneous polymerization (within 10 seconds) of the protomeric form giving rise to filamentous structures 70 to 100 A in width and up to 0.5// in length (Fig. IB, reference [176]). 

Fig. 7. Characteristics of the protomeric and polymeric forms of avian liver acetyl-CoA carboxylase [128,176,177,180].

The citrate concentration of whole liver in the fed state is approximately 0.6 mM [209,213]. This concentration is two orders of magnitude greater than the dissociation constant [180] found for citrate with the avian liver acetyl-CoA carboxylase K = 0.003 mAf) but somewhat lower than the activator constant [128,178] for citrate = 2 to 3 mM). 

Malonyl-CoA is a potent inhibitor of the avian liver acetyl-CoA carboxylase [94,177], exhibiting a Ki of about 10" M. It has been ascertained that inhibition by malonyl-CoA is competitive, with respect to both acetyl-CoA and tricarboxylic acid activator [177]. While competitive inhibition by malonyl-CoA with respect to acetyl-CoA is of the classical type, the competitive relationship between malonyl CoA and isocitrate is best interpreted in terms of the protomer polymer equilibrium shown in Fig. 7. Citrate and isocitrate cause a shift in the equilibrium toward the catalytically active polymeric form, while malonyl-CoA is known to promote depolymerization [177,180], thereby shifting the equilibrium toward the catalytically less active protomer. Carboxylation of the enzyme by malonyl-CoA to form enzyme-C02 produces a strained conformation which favors depolymerization as discussed previously (see Section V, C, 1, c). The fact that the capacity of the carboxylase to generate malonyl-CoA approximately equals the capacity of the synthetase to incorporate malonyl-CoA into long-chain fatty acids suggests that circumstances... 

There is abundant evidence indicating that a natural hydrophobic inhibitor of acetyl-CoA carboxylase is present in crude enzyme extracts of liver and adipose tissue [128,129,182,192,236-238]. The activating effect of (+)-palmityl carnitine on fatty acid synthesis in crude liver extracts and on impure acetyl-CoA carboxylase preparations has tentatively been ascribed to the displacement of hydrophobic inhibitors such as fatty acids or fatty acyl-CoA derivatives [129,182,192,236-238]. Inhibition of rat liver acetyl-CoA carboxylase by added palmityl-CoA can be reversed in part by (+)-palmityl carnitine [236], but not by citrate. This activating effect does not appear to be specific with respect to (+)-palmityl carnitine in that cetyl trimethylammonium ion is also effective [192]. Furthermore, impure preparations of acetyl-CoA carboxylase from adipose tissue or rat liver are markedly activated by serum albumin [123,129,238] or extensive dilution of the enzyme preparation prior to assay [129,182]. On the other hand, none of these agents [(+)-palmityl carnitine, serum albumin, or dilution], which activate the impure carboxylase, have an activating effect on the homogeneous acetyl-CoA carboxylases from adipose tissue or liver [129,182, 239]. It is evident that an inhibitory substance, apparently hydrophobic in nature, is removed either by purification of the enzyme or by the agents or treatments mentioned above. 

It has been reported that, like liver acetyl-CoA carboxylase, both the liver and yeast fatty acid synthetases are inhibited by low concentrations (0.5 to 5 X 10 71/) of long-chain fatty acyl-CoA derivatives, the longer-chain derivative producing greater inhibition [226,246,247]. In the case of the yeast synthetase, inhibition by long-chain acyl-CoA derivatives was competitive with respect to acetyl-CoA and NADPH. For the same reasons alluded to earlier in the discussion of the inhibition of acetyl-CoA carboxylase by fatty acyl-CoA derivatives, some caution must be exercised in interpreting the effect of these potent inhibitors (see Section V, C, 2). 

In addition to being the principal substrate for the carboxylase, acetyl-CoA also appears to have a role in its regulation. Studies of the partial reactions of rat liver acetyl-CoA carboxylase by Lynen et al. 

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