Acetoacetyl-CoA synthetase

Fig. 1. The metabolic cycle for the synthesis and degradation of poly(3HB). (1) 3-ketothiolase (2) NADPH-dependent acetoacetyl-CoA reductase (3) poly(3HB) synthase (4) NADH-dependent acetoacetyl-CoA reductase (5), (6) enolases (7) depolymerase (8) d-(-)-3-hydroxybutyrate dehydrogenase (9) acetoacetyl-CoA synthetase (10) succinyl-CoA transferase (11) citrate synthase (12) see Sect. 3...

Most tissues oxidize the acetyl-CoA produced during P-oxidation to C02 and water via the TCA cycle. During fasting, however, the liver utilizes the intermediates of the TCA cycle as gluconeogenic substrates. Under these conditions, the Ever converts acetyl-CoA to ketone bodies (acetoacetate and P-hydroxybutyrate) (Figure 32-5). Most other peripheral tissues can oxidize ketone bodies by the pathway shown in the figure. After entering the mitochondria, acetoacetate reacts with succinyl-CoA to form acetoacetyl-CoA, a reaction that is catalyzed by 3-oxoacid-CoA transferase. Alternatively, acetoacetyl-CoA is formed by direct activation of acetoacetate by the enzyme acetoacetyl-CoA synthetase. Acetoacetyl-CoA is then cleaved to form two molecules of acetyl-CoA by acetoacetyl-CoA thiolase.As noted earlier in... 

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. 

Because of the poor equilibrium position of acetoacetyl-CoA synthetase, they tested the effect of the combined presence of acetoacetyl-CoA synthetase and HMG-CoA synthetase on the equilibrium between acetyl-CoA and HMG-CoA. At equilibrium slightly more than half of the acetyl-CoA had been converted to product. The position of the equilibrium is governed by the expression... 

The existence in the brain cytoplasm of acetoacetyl-CoA synthetase and acetoacetyl-CoA thiolase makes unnecessary the existence of a transmitochondrial membrane transport of acetyl CoA for lipid synthesis. We have found a clear inhibitory effect on the activity of the thiolase by several metabolites of phenylalanine. Also the hydroxybutyrate dehydrogenase of the suckling rat brain is inhibited by these metabolites. These effects are interesting in relation to the pathophysiology of mental retardation in phenylketonuria, in which the concentration of such metabolites are considerably increased. It is worth noting that this inhibition can influence the rate of synthesis of fatty acids and cholesterol (precursors of myelin), especially during brain development. 

The copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-co-PHV) produced by A eutrophus has generated more interest than poly-(R)-3-hydroxybutyrate (PHB) homopolymer. Since these bacterial polyesters are biodegradable thermoplastics, their mechanical and physical properties have received much attention. PHB is a relatively stiff and brittle material because of its high crystallinity. However, the physiochemi-cal and mechanical properties of [P(HB-HV)] vary widely and depend on the molar percentage of 3-hydroxyvalerate (HV) in the copolymer (4,5) as shown inTable 1. Propionic acid is converted by a synthetase to propionyl-CoA, and the biosynthetic P-ketothiolase catalyzes the condensation of propionyl-CoA with acetyl-CoA to 3-ketovaleryl-CoA by the acetoacetyl-CoA reductase. The hydroxyvaleryl moiety is finally covalently linked to the polyester by the PHA synthase (6). 

Tissues other than liver utilize ketone bodies by first reoxidizing /3-d-hydroxybutyrate to acetoacetate (see Figure 19.10) and then converting acetoacetate to acetoacetyl-CoA. The latter occurs via a mitochondrial thiophorase reaction [Equation (19.8)] or a cytosolic acetoacetate CoA synthetase reaction [Equation (19.9)] ... 

Brief incubation of the enzyme at 0°C with radioactive acetyl-CoA led to the formation of acyl enzyme which could be isolated by chromatography on Sephadex [24]. The enzyme-substrate complex was then reacted with acetoacetyl-CoA with the concomitant formation of HMG-CoA. Further studies indicated that the functional group on the enzyme that accepted the acetyl residue was a cysteine sulfhydryl. 4 -Phosphopantetheine is known to accept acyl residues, but it was not found in this protein. The stoichiometry for acetylation was 0.7 acetyl groups per mole of enzyme since it is a dimeric protein with apparently identical subunits, this observation is surprising. Thus, it is possible that the subunits perform different functions for example, one could be regulatory. It is interesting to note that both the thiolase and HMG-CoA synthetase utilize acyl enzyme intermediates in their catalytic mechanisms. 

In a later publication, another enzyme-bound intermediate involved in the reaction mechanism of HMG-CoA synthetase was reported [26]. On the assumption that acetylation of the enzyme by acetyl-CoA was the initial step in the reaction sequence, they felt that condensation with the second substrate, acetoacetyl-CoA,... 

Steroids are members of a large class of lipid compounds called terpenes. Using acetate as a starting material, a variety of organisms produce terpenes by essentially the same biosynthetic scheme (Fig. 8). The self-condensation of two molecules of acetyl coenzyme A (CoA) forms acetoacetyl CoA. Condensation of acetoacetyl CoA with a third molecule of acetyl CoA, then followed by an NADPH-mediated reduction of the thioester moiety produces mevalonic acid [150-97-0] (72). Phosphorylation of (72) followed by concomitant decarboxylation and dehydration processes produce isopentenyl pyrophosphate. Isopentenyl pyrophosphate isomerase establishes an equilibrium between isopentenyl pyrophosphate and 3,3-dimethylallyl pyrophosphate (73). The head-to-tail addition of these isoprene units forms geranyl pyrophosphate. The addition of another isopentenyl pyrophosphate unit results in the sesquiterpene (C15) famesyl pyrophosphate (74). Both of these head-to-tail additions are catalyzed by prenyl transferase. Squalene synthetase catalyzes the head-to-head addition of two achiral molecules of famesyl pyrophosphate, through a chiral cyclopropane intermediate, to form the achiral triterpene, squalene (75). 

Acetoacetate can be activated to acetoacetyl CoA in the cytosol by an enzyme similar to the acyl CoA synthetases. This acetoacetyl CoA can be used directly in cholesterol synthesis. It also can be cleaved to two molecules of acetyl CoA by a cytosolic thiolase. Cytosolic acetyl CoA is required for processes such as acetylcholine synthesis in neuronal cells. 

Building blocks for cholesterol include acetate, acetyl-CoA and acetoacetyl CoA. There are many sources of these metabolites in the body including the tricarboxylic acid cycle (TCA) but also microbial-derived acetate, the main end-product of carbohydrate fermentation in the colon which is converted into acetyl-CoA by acetyl coenzyme A synthetase 1 (AceCSl) in the cytosol. Acetate may also be taken up by mitochondria and converted into acetyl-coA by AceCS2 for respiration through the TCA, especially under ketogenic or fasting conditions. In... 

Very recently interest has focused on still earlier control sites in the sterol pathway. According to the important findings of Lane e. al. (3), beta-ketoacyl thiolase and HMG-CoA synthetase occur in the cytoplasm as well as in the mitochondria. A cytoplasmic provision of acetoacetyl-CoA and HMG-CoA would first of all make sterol synthesis autonomous and independent of the mitochondrial supply of these precursors. At the same time HMG-CoA would lose its status as the common intermediate in both keto-genesis and cholesterol synthesis. It seems significant in this context that the citric acid analogue, hydroxycitrate, not only blocks hepatic cholesterogenesis but also inhibits ATP citrate lyase, the enzyme that supplies extramitochondrial acetyl-CoA. 

Acetyl-CoA ACP transacylase is a thiolactomycin-sensitive enzyme which catalyzes what has often been regarded as the slowest of all the partial reactions. However, the recent observation that in spinach leaves and all other plant tissues examined there is an acetoacetyl-ACP synthetase which bypasses this reaction (and which has much higher activity) sheds doubt on the physiological importance of acetyl-CoA ACP transacylase. The acetoacetyl-ACP synthetase, sometimes called the short-chain condensing enzyme, is cerulenin insensitive and can, therefore, be differentiated easily from the )8-ketoacyl-ACP synthetase I, which is the main condensing enzyme. The latter has been purified recently to homogeneity for the first time. " ... 

Very recently a third condensing enzyme has been reported in E. coli. This condensing enzyme is distinctly different from the other -ketoacyl-ACP synthetases in E. coli in that it is (a) cerulenin-insensitive (b) specific for very short chain acyl-ACPs and (c) prefers acetyl-CoA over acetyl-ACP. It has been termed acetoacetyl-ACP synthetase. A similar enzyme has... 

The first two steps involve condensation reactions catalysed by a thiolase and hydroxymethylglutaryl-CoA(HMG-CoA) synthetase. Both enzymes are soluble and the first reaction is driven to completion by rapid removal of acetoacetyl-CoA by the second step (Figure 7.16). 

HMG-CoA synthetase has been studied in considerable detail and the reaction mechanism defined. It shows a very high degree of specificity with regard to the stereochemistry of the acetoacetyl-CoA substrate and the condensation proceeds by inversion of the configuration of the hydrogen atoms of acetyl-CoA. In addition to cytosolic HMG-CoA synthetase, a second synthetase is found in mitochondria. Not only has this been shown to be a different protein, the HMG it forms has a different function. Whereas HMG in the cytosol is destined for mevalonate formation, that in mitochondria is broken down by HMG-CoA lyase to yield acetyl-CoA and acetoacetate (section 3.3.1). 

Investigations of liver biopsy material for enzymes involved in lactic acidosis and ketogenesis showed normal activites of pyruvate carboxylase, citrate synthetase, isocitrate dehydrogenase, glutamate-pyruvate transaminase, reduced activity of fructose 1,6-bisphosphatase and notably reduced activity of cytosolic acetoacetyl-CoA thiolase. The latter was found to be due to altered kinetic properties of the enzyme and this was confirmed in cultured skin fibroblasts, the enzyme being much more sensitive to coenzyme A inhibition than the normal enzyme. Activity of succinyl-CoA 3-keto acid-CoA transferase was not reported. 

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