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Studies on acetoacetate formation Caldwell, Ian Carl


In recent years, two mechanisms have been proposed for the enzymatic formation of acetoacetate by liver extracts. One of these, the "HMG-CoA cycle", involves the condensation of acetyl-CoA and acetoacetyl-CoA to form β-hydroxy- β-methylglutaryl-CoA (HMG-CoA) via the action of the HMG-CoA condensing enzyme, with the release of free coenzyme A (CoASH) (reaction 1). Acetyl-CoA + acetoacetyI-CoA + H₂O⇆ HMG-CoA + CoASH (1) followed by cleavage of the HMG-CoA to acetyl-CoA and free acetoacetate, via the action of the HMG-CoA cleavage enzyme (reaction 2). HMG-CoA⇆ acetoacetate + acetyl-CoA (2) The second mechanism which has been proposed involves a direct deacylation of acetoacetyl CoA through the action of a specific acetoacetyl-CoA thioesterase (reaction). Acetoacetyl-CoA + H₂O→ acetoacetate + CoASH (3) Evidence is presented which indicates acetoacetate formation by a soluble enzyme system from bicarbonate extracts of whole beef liver proceeds largely, if not exclusively, via HMG-CoA (reactions 1 and 2). Both the HMG-CoA condensing and cleavage enzymes have been partially purified from beef liver bicarbonate extracts, each free of contamination by the other, in good yields. The level of activity of these two enzymes is sufficiently high to account for all the acetoacetate formed by liver tissue. The possibility that the specific acetoacetyl-CoA thioesterase may play a minor role in the enzymatic synthesis of acetoacetate is also discussed. The intracellular and tissue localization of the enzymes of acetoacetate formation is also discussed. In liver homogenates, most, if not all, of the acetoacetate-synthesizing activity appears to be associated with the mitochondrion. Evidence is also presented that the primary reason for the inability of extrahepatic tissue preparations to catalyze the accumulation of acetoacetate may be the lack of one of the enzymes involved, i.e., the HMG-CoA condensing enzyme, and not merely further metabolic degradation of acetoacetate, as has generally been assumed. An enzyme fraction in chicken liver extracts which inhibits the in vitro formation of acetoacetate by chicken liver homogenates has also been studied. Evidence is presented that this enzyme fraction exerts its effect through the inactivation of coenzyme A. Preliminary observations indicate that this enzyme may be a 3’-nucleotidase, removing the 3’- phosphate of coenzyme A, forming dephosphocoenzyme A. The occurrence of a highly active β-hydroxybutyryl dehydrogenase in extracts of dry culture of C. kluyveri has been noted. This enzyme differs from the similar enzyme reported in mammalian tissues, in that it is very specific for triphosphopyridine nucleotide, and is virtually inactive with diphosphopyridine nucleotide (DPN) (reaction). Acetoacetyl-CoA + TPNH + H⁺⇆β - hydroxybutyryl-CoA (4) + TPN⁺

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