Acetoacetyl-coenzyme A thiolase

Vollack, K.U. and Bach, T.J. (1996) Cloning of a cDNA encoding cytosolic acetoacetyl-coenzyme A thiolase from radish by functional expression in Saccharomyces cere-visiae. Plant Physiol., Ill, 1097-107. 

The incorporation of malonate into mevalonic acid and steroids has been investigated further. Experiments with normal and tumorous rats have demonstrated the previously unsuspected fact that the S-methyl group of methionine is incorporated into cholesterol and cholest-7-en-3jS-ol. Some of the enzymes involved in mevalonate synthesis have been isolated. Yeast acetoacetyl coenzyme A thiolase (EC 2.3.1.9) has a molecular weight of about 190 000 and 3-hydroxy-3-methyl glutaryl coenzyme A synthetase (EC 4.1.3.5) a molecular weight of 130 000. Rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) used only [4/ - H]NADPH in the formation of mevalonic acid with incorporation of two tritium atoms (at Hp and Hp) (see Scheme 1). 

Fukao T, et al. Molecular basis of beta-ketothiolase deficiency mutations and polymorphisms in the human mitochondrial acetoacetyl-coenzyme A thiolase gene. Hum Mutat. 1995 5(2) 113-20. 

MOLECULAR CLONING OF RADISH ACETOACETYL-COENZYME A THIOLASE BY GENETIC COMPLEMENTATION OF A YEAST MUTANT... 

Middleton, B. (1973b), The acetoacetyl-coenzyme A thiolases of rat brain and their relative activities during postnatal development. Biochem. J. 132,731. 

After the activation to its coenzyme A ester of acetoacetate by succinyl-CoA 3-oxoacid transferase, the acetoacetyl-coenzyme A must be cleaved into acetyl-CoA by thiolase. There are three thiolase enzymes one cytosolic enzyme and two mitochondrial enzymes (distinguished by the laboratory property of one being activated by potassium and the other is not). The main enzyme only cleaves acetoacetyl-CoA and provides a baseline thiolase activity. The other enzyme cleaves both acetoacetyl-CoA and... 

An example in which reactivity of both the attacking nucleophile and the electrophilic acceptor is dependent on the special character of acyl thioesters is in the condensation of two acetyl coenzyme A units to form acetoacetyl coenzyme A. This is the reverse of the thiolase reaction... 

This enzyme [EC 2.3.1.9], also known as thiolase, transfers an acetyl group from one acetyl-CoA molecule to another to form free coenzyme A and acetoacetyl-CoA. 

In extraliepatic tissues, d-/3-hydroxybutyrate is oxidized to acetoacetate by o-/3-hydroxybutyrate dehydrogenase (Fig. 17-19). The acetoacetate is activated to its coenzyme A ester by transfer of CoA from suc-cinyl-CoA, an intermediate of the citric acid cycle (see Fig. 16-7), in a reaction catalyzed by P-ketoacyl-CoA transferase. The acetoacetyl-CoA is then cleaved by thiolase to yield two acetyl-CoAs, which enter the citric acid cycle. Thus the ketone bodies are used as fuels. 

FIGURE 9.2 Physiology of ABE fermentation metabolism of Clostridium acetobutylicum with the respective enzymes and products. CoA, coenzyme A Ldh, lactate dehydrogenase Pdc, pyruvate decarboxylase Pfor, pyruvate ferredoxin oxidoreductase Fdred, ferredoxin reduced Thl, thiolase Hbd, p-hydroxybutyryl-CoA dehydrogenase Crt, crotonase Bed, butyryl-CoA dehydrogenase Etf, electron transfer flavoprotein Pta, phosphotransacetylase Ack, acetate kinase Ptb, phosphotransbutyrylase Buk, butyrate kinase Ctf A/B, acetoacetyl-CoA acyl-CoA transferase Adc, acetoacetate decarboxylase AdhE, aldehyde/alcohol dehydrogenase Bdh, butanol dehydrogenase. 

In a Claisen condensation catalyzed by the enzyme thiolase, acetyl-CoA is converted to its enolate anion, which then adds to the carbonyl group of a second molecule of acetyl-CoA to give a tetrahedral carbonyl addition intermediate. Collapse of this intermediate by elimination of coenzyme A anion (CoAS ) gives acetoacetyl-CoA.Subsequentproton transfer to coenzyme Aanion gives coenzyme A. The mechanism of this reaction is identical to that of the Claisen condensation (Section 19.3A). 

The first reaction consists of the condensation of two acetyl coenzyme A (acetyl-CoA) molecules into acetoacetyl-CoA by 3-ketoacylCoA thiolase. The second reaction is the reduction of acetoacyl-CoA to (R)-3-hydroxybutyiyl-CoA by an NADPH-dependent acetoacetyl-CoA dehydrogenase. Lastly, the (R)-3-hydroxybutyryl-co-A monomers are polymerized into PHB by P(3HB) polymerase. 

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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