Acetoacetate from leucine

HMG-CoA is also synthesized in mitochondria by the same sequence of reactions but yields the ketone bodies acetoacetate, D(—)- 8-hydroxybutyrate, and acetone (Figure 19-10). Mitochondrial HMG-CoA also arises from oxidation of leucine (Chapter 17), which is keto-genic. Although HMG-CoA derived from leucine is not utilized in mevalonate synthesis, the carbon of leucine can be incorporated into cholesterol by way of acetyl-CoA. Thus, two distinct pools of HMG-CoA exist one... 

Echinulin.— The mould metabolite echinulin (126) and its relatives have been the subject of some elegant biosynthetic experiments leading to a clear picture of the way they are elaborated." Echinulin has recently been used as a monitor for the stereochemistry involved in the catabolism of leucine to mevalonic acid " label from the leucine was expected at specific sites in the mevalonate-derived iso-prenoid units of (126). In the event, extensive scrambling of label was observed from the two enantiomers of leucine chirally labelled with "C at C-3. Some selectivity in the labelling was detected, which was interpreted as indicating that carriage of label from leucine to mevalonate had occurred via acetoacetate. 

More precise information on the pathway of leucine catabolism was obtained from studies on the formation of ketone bodies in liver slices incubated with and C Mabeled leucine and isovaleric acid. In these experiments it was found that leucine-3-C yielded acetoacetate in which the label was virtually all contained in the methyl and methylene carbons, and to approximately the same extent in each of these. Only a trace of radioactivity was found in the carboxyl carbon. On incubation with leucine-4-Ci the label occurred solely in the carbonyl group. This suggested that the isopropyl group of the amino acid had been directly converted to acetone. The over-all conclusion was that the isopropyl group forms acetone, and carbons 2 and 3 of the amino acid yield a 2-car-bon fragment which can condense to acetoacetate. The acetoacetate formed from leucine-4-C was not symmetrically labeled, the isotope being present only in the carbonyl carbon. 

The formation of acetoacetate from the isopropyl moiety of leucine, then, appears to follow the course depicted in reactions 3 to 5 of Fig. 5. It has been noted that a greater amount of ketone bodies was produced from the D- or Dii-leucine than from the n-isomer. Since both optical isomers yield the same keto acid upon oxidative deamination, their carbon chains would not be expected to meet with different catabolic fates. The difference, it would appear, is the result of a difference in the rates of deamination of the D- and n-isomers. 

Methylcrotonyl CoA Carboxylase Methylcrotonyl CoA carboxylase catalyzes the conversion of methylcrotonyl CoA, arising from the catabolism of leucine, to methylglutaconyl CoA. This in turn undergoes hydroxy-lation catalyzed by crotonase, yielding hydroxymethyl-glutaryl CoA, which is cleaved to acetyl CoA and acetoacetate. 

C. HMG CoA is not formed from glutamic acid, but from acetyl CoA and acetoacetyl CoA. It is also formed by degradation of leucine in muscle. It is cleaved to form acetyl CoA and the ketone body acetoacetate. It is reduced to mevalonic acid in cholesterol biosynthesis. 

Acetyl CoA serves as a common point of convergence for the major pathways of fuel oxidation. It is generated directly from the (3-oxidation of fatty acids and degradation of the ketone bodies (3-hydroxybutyrate and acetoacetate (Fig. 20.14). It is also formed from acetate, which can arise from the diet or from ethanol oxidation. Glucose and other carbohydrates enter glycolysis, a pathway common to all cells, and are oxidized to pyruvate. The amino acids alanine and serine are also converted to pyruvate. Pyruvate is oxidized to acetyl CoA by the pyruvate dehydrogenase complex. A number of amino acids, such as leucine and isoleucine are also oxidized to acetyl CoA. Thus, the final oxidation of acetyl CoA to CO2 in the TCA cycle is the last step in all the major pathways of fuel oxidation. 

In mammalian cells, HMG-CoA is a substrate for several enzymes involved in metabolic processes, including cholesterogenesis, ketogenesis, and leucine metabolism. HMG-CoA lyase (EC 4.1.3.4) catalyzes the formation of the ketone body acetoacetate and acetyl-CoA from HMG-CoA. Acetoacetate is regarded as a transportable form of acetyl-CoA and plays an important role in metabolism during fasting (85). The enzyme is localized in the mitochondria and is also involved in leucine metabolism. Recently, this enzyme has been partially purified from radish (86) and from C. roseus... 

Incorporation of leucine into the C isoprene units of echinulin (112) through catabolism to mevalonate is thought to proceed via acetoacetate cf. ref. 9. Loss of C-2 of leucine is required in this pathway, which is supported by loss of label from [2- C, 5- H]leucine on incorporation into echinulin (also some loss of tritium, measured in another experiment). ... 

Stereospecific a,/i-desaturation of isovalerylcoenzyme A derived from the oxidative degradation of leucine would yield a )8-methyl crotonylco enzyme A with retention of the pro-R/pro-S-laheled leucine-derived stereochemistry as shown. Subsequent cleavage of /l-hydroxy-)3-methylglutarylcoenzyme A into acetyl CoA and acetoacetate followed by intact incorporation of the acetoacetate into mevalonate can account for the observed labeling propensities. 

An interesting and unusual synthesis of racemic leucine (Leu, L) was carried out as shown in Scheme 12.34 and reported early in the twentieth century. As shown, 2-methyl-l-propanol was converted to the corresponding iodide with hydrogen iodide (HI) and the primary alkyl iodide reacted with sodium salt derived from methyl acetoacetate to yield 5-methyl-3-carboxymethyl-2-hexanone. This, treated with nitrosyl sulfate (0=N-0S03H), yields the decarbonylated methyl 5-methyl-pentanoate-2-oxime. Reduction of the latter produced racemic leucine (Leu, L). 

An interesting finding resulting from the study of the enzymes that participate in the conversion of leucine to acetoacetate is that the j3-methyl-crotonyl CoA carboxylase, the enzyme that forms /9-hydroxy-/8-methyl-glutaryl CoA, is biotin-dependent 104)-... 

The nature of the 3-carbon fragment from the isopropyl group for the case where n = odd has been provided from isotope studies with leucine and isovaleric acid. When leucine labeled in the branching carbon (y-carbon) is incubated with rat liver slices, the isotope is found only in the carbonyl carbon of acetoacetate (acetone fraction). Isovalerate, in which the methyl carbons of the isopropyl group are labeled, gives rise to acetoacetate with isotope only in the a- and y-carbons of acetoacetate (acetone fraction). These experiments indicate that the 3-carbon fragment from the isopropyl group of leucine or isovaleric acid is closely related to acetone. 

In the metabolism of L-leucine, the isovaleryl-CoA produced by the oxidative decarboxylation step is further metabolized by a series of enzyme-catalysed steps to acetoacetate and acetyl-CoA and thence into the tricarboxylic acid cycle. Specific enzyme deficiencies at every stage of this metabolic pathway are known and are described in Section 10.3. In contrast, only one disorder of L-isoleucine metabolism subsequent to the oxidative decarboxylation step has been recognized (Section 10.4), and no disorders of the L-valine pathway from isobutyryl-CoA have been described. This may be due to their relative rarity but possibly also to greater difficulty in their detection. The metabolism of valine and leucine is, however, of particular interest in the organic acidurias, since both are major precursors of propionyl-CoA and methylmalonyl-CoA, defects in the metabolism of which lead to propionic acidaemia and methylmalonic aciduria (Chapter 11). 

---