Acetoacetic acid, activation conversion

The conversion of acetoacetic acid to hydroxybutyric acid restitutes the 4-carbon compounds to the general metabolism because the liver contains enzymes that will activate jS-hydroxybutyric acid to yield j8-hydroxybutyric CoA, whereas acetoacetic acid cannot be acylated by the liver. 

Hydroxy- 9-methylglutaryl CoA further yields acetyl CoA and acetoacetic acid, as was shown earlier by Coon et cU. (I48). In biotin deficiency the carboxylation reaction does not occur. It was shown by Lynen et al. that the actual carboxylation is preceded by the enzymic dehydration (rf jS-hydroxyisovaleryl CoA to /8-methylcrotonyl CoA, which is the true substrate for the entry of CO2. TTiis occurs at the expense of the hydrolysis of the terminal P04 of ATP. The unsaturated intermediate is then saturated by the addition of H2O to yield the final product. The critical step of this carboxylation is the conversion of CO2 to a reactive form. The analogy of the biochemical activation of other substances through an acyl adenylate type of compound did not fit CO2 activation. The final mechanism of the activation of CO2 was derived from the discovery that the carboxylase enzyme was a biotin-protein. This observation explains earlier work 149) which indicated that biotin is a cofactor of the fatty acid-synthesizing enzyme system. When the purified carboxylase was incubated with P and ATP an exchange reaction of phosphate occurred, which was inhibited by avidin, a protein which specifically binds biotin. This indicated that the primary reaction in CO2 fixation is the combination of ATP with the biotin-protein enzyme to yield ADP biotin-protein -f P. The active CO2 is then the product of an exchange reaction between ADP and C02 which is finally attached to the biotin-protein complex. 

The procedure described here for compound 1 is a scaleup of a published method.6 Phase-transfer catalysis7 and concentrated alkali are used to effect a one-pot conversion of diethyl malonate to the cyclopropane diacid, which is easily obtained by crystallization. Apparently alkylation of the malonate system occurs either at the diester or monocarboxylate, monoester stage since the method fails when malonic acid itself is used as the starting material. This method of synthesizing doubly activated cyclopropanes has been extended to the preparation of 1-cyanocyclopropanecar-boxylic acid (86%) by the use of ethyl cyanoacetate and 1-acetyl-cyclopropanecarboxylic acid (69%) by use of ethyl acetoacetate.6... 

The actual pKa value of an active site catalytic group will be influenced by the particular microenvironment of the active site, which could raise or lower the pKa. For example, the enzyme acetoacetate decarboxylase contains an active site lysine residue that forms an imine link with its substrate its pKa value was found to be 5.9, which is much less than the expected value of 9. Adjacent to this residue in the active site is a second lysine residue, which in protonated form destabflizes the protonated amine and, therefore, reduces the pKa. Conversely, aspartic acid or glutamic acid residues that are positioned in hydrophobic active sites can have increased pKa values near 7 because the anionic form of the side chain is destabilized. 

The liver also plays a central role in lipid metabolism. When excess fuel is available, the liver synthesizes fatty acids. These are used to produce triglycerides that are transported from the liver to adipose tissues by very low density lipoprotein (VLDL) complexes. In fact, VLDL complexes provide adipose tissue with its major source of fatty acids. This transport is particularly active when more calories are eaten than are burned During fasting or starvation conditions, however, the liver converts fatty acids to acetoacetate and other ketone bodies. The liver cannot use these ketone bodies because it lacks an enzyme for the conversion of acetoacetate to acetyl CoA. Therefore the ketone bodies produced by the liver are exported to other organs where they are oxidized to make ATP. 

Wiesenborn etal. [64] purified the CoA transferase of C. acetohutylicum ATCC 824 and determined values for acetate (1200 mM) and butyrate (660 mM). In C. acetohutylicum, intracellular concentrations of acetate and butyrate are generally much lower than the apparent values and do not exceed 300 and 700 mM, respectively. Enzyme assays also showed that acetate and butyrate conversion was suppressed by physiological levels of acetone (200 mM), butanol (200 mM), acetoacetate (20 mM), and acetyl-CoA (1 mM). The activity of Adc is equally dependent on concentration of acetate in the medium [65]. Conversion of organic acids by CoA transferase was not observed until induction of Adc. Overall, the... 

The utilization of acetoacetate is controlled by the activity of the citric acid cycle. The reaction of acetoacetate succinyl CoA transferase provides an alternative to the reaction of succinyl CoA synthase (see Figure 5.18), and there will only be an adequate supply of succinyl CoA to permit conversion of acetoacetate to acetoacetyl CoA as long as the rate of citric acid cycle activity is adequate. 

Fatty acid oxidation can be terminated in either of two ways. Acetoacetyl CoA can either be cleaved to two molecules of acetyl CoA which condense with oxalacetate to form citrate, or it can be deacylated to acetoacetate by a deacylase specific for d-keto butyryl CoA. - In kidney and heart muscle there is no accumulation of acetoacetate, whereas in liver acetoacetate is formed in preference to citrate. The non-accumulation of acetoacetate in tissues other than liver probably is referable to the following circumstances. All tissues but liver contain activating enzymes which catalyze the conversion of acetoacetate to acetoacetyl CoA. Thus any acetoacetate formed by deacylation is thrust back as it were into the metabolic wheel. In liver deacylation is not opposed by this reactivation of acetoacetate. Hence acetoacetate accumulates only in liver. 

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