Ketoacyl CoA

The third reaction of this cycle is the oxidation of the hydroxyl group at the /3-position to produce a /3-ketoacyl-CoA derivative. This second oxidation reaction is catalyzed by L-hydroxyacyl-CoA dehydrogenase, an enzyme that requires NAD as a coenzyme. NADH produced in this reaction represents metabolic energy. Each NADH produced in mitochondria by this reaction drives the synthesis of 2.5 molecules of ATP in the electron transport pathway. L-Hydroxyacyl-... 

Step 3 of Figure 29.3 Alcohol Oxidation The /3-hydroxyacyl CoA from step 2 is oxidized to a /3-ketoacyl CoA in a reaction catalyzed by one of a family of L-3-hydroxyacyl-CoA dehydrogenases, which differ in substrate specificity according to the chain length of the acyl group. As in the oxidation of sn-glycerol 3-phosphate to dihydroxyacetone phosphate mentioned at the end of Section 29.2, this alcohol oxidation requires NAD+ as a coenzyme and yields reduced NADH/H+ as by-product. Deprotonation of the hydroxyl group is carried out by a histidine residue at the active site. 

Step 4 of Figure 29.3 Chain Cleavage Acetyl CoA is split off from the chain in the final step of /3-oxidation, leaving an acyl CoA that is two carbon atoms shorter than the original. The reaction is catalyzed by /3-ketoacyl-CoA thiolase and is mechanistically the reverse of a Claisen condensation reaction (Section 23.7). In the forward direction, a Claisen condensation joins two esters together to form a /3-keto ester product. In the reverse direction, a retro-Claisen reaction splits a /3-keto ester (or /3-keto thioester) apart to form two esters (or two thioesters). 

The retro-Claisen reaction occurs by initial nucleophilic addition of a cysteine -SH group on the enzyme to the keto group of the /3-ketoacyl CoA to yield an alkoxide ion intermediate. Cleavage of the C2-C3 bond then follows, with expulsion of an acetyl CoA enolate ion. Protonation of the enolate ion gives acetyl CoA, and the enzyme-bound acyl group undergoes nucleophilic acyl substitution by reaction with a molecule of coenzyme A. The chain-shortened acyl CoA that results then enters another round of tire /3-oxidation pathway for further degradation. 

Uchicda, Y., Izai, K., Orii, T., Hashimoto, T. (1992). Novel fatty acid p-oxidation enzymes in rat liver mitochondria. II. Purification and properties of enoyl-coenzyme A (CoA) hydratase/3-hy-droxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein. J. Biol. Chem. 267, 1034-1041. 

Inherited defects in the enzymes of (3-oxidation and ketogenesis also lead to nonketotic hypoglycemia, coma, and fatty hver. Defects are known in long- and short-chain 3-hydroxyacyl-CoA dehydrogenase (deficiency of the long-chain enzyme may be a cause of acute fetty liver of pr nancy). 3-Ketoacyl-CoA thiolase and HMG-CoA lyase deficiency also affect the degradation of leucine, a ketogenic amino acid (Chapter 30). 

The 3-ketothiolase has been purified and investigated from several poly(3HB)-synthesizing bacteria including Azotobacter beijerinckii [10], Ral-stonia eutropha [11], Zoogloea ramigera [12], Rhodococcus ruber [13], and Methylobacterium rhodesianum [14]. In R. eutropha the 3-ketothiolase occurs in two different forms, called A and B, which have different substrate specificities [11,15]. In the thiolytic reaction, enzyme A is only active with C4 and C5 3-ketoacyl-CoA whereas the substrate spectrum of enzyme B is much broader, since it is active with C4 to C10 substrates [11]. Enzyme A seems to be the main biosynthetic enzyme acting in the poly(3HB) synthesis pathway, while enzyme B should rather have a catabolic function in fatty-acid metabolism. However, in vitro studies with reconstituted purified enzyme systems have demonstrated that enzyme B can also contribute to poly(3HB) synthesis [15]. 

In studies of R. ruber, only one active enzyme has been found, although the possibility that a second unstable enzyme exists has not been excluded. Its activity was greatest with acetoacetyl-CoA, and two-thirds lower with 3-keto-valeryl-CoA. Activity was also found with C6 to C8 3-ketoacyl-CoAs [13]. 

Fatty acids are degraded by two-carbon units in a reverse manner analogous to their biosynthesis. The acyl-CoAs are first dehydrogenated to a,(3-unsaturated acyl-CoA, and then hydrated to (3-hydroxyacyl-CoA, followed by oxidation to (3-ketoacyl-CoA. The C-C bond between C-2 and C-3 of the latter compound is broken by a free CoA molecule via thiolysis to form an acyl-CoA that is two carbons shorter and acetyl-CoA. Unlike fatty acid biosynthesis, each step of the (3 oxidation of fatty acids is... 

This enzyme [EC 2.3.1.16], also known as 3-ketoacyl-CoA thiolase, transfers an acyl group from an acyl-CoA to acetyl-CoA to form free coenzyme A and 3-oxoa-cyl-CoA. 

Desaturation of alkyl groups. This novel reaction, which converts a saturated alkyl compound into a substituted alkene and is catalyzed by cytochromes P-450, has been described for the antiepileptic drug, valproic acid (VPA) (2-n-propyl-4-pentanoic acid) (Fig. 4.29). The mechanism proposed involves formation of a carbon-centered free radical, which may form either a hydroxy la ted product (alcohol) or dehydrogenate to the unsaturated compound. The cytochrome P-450-mediated metabolism yields 4-ene-VPA (2-n-propyl-4pentenoic acid), which is oxidized by the mitochondrial p-oxidation enzymes to 2,4-diene-VPA (2-n-propyl-2, 4-pentadienoic acid). This metabolite or its Co A ester irreversibly inhibits enzymes of the p-oxidation system, destroys cytochrome P-450, and may be involved in the hepatotoxicity of the drug. Further metabolism may occur to give 3-keto-4-ene-VPA (2-n-propyl-3-oxo-4-pentenoic acid), which inhibits the enzyme 3-ketoacyl-CoA thiolase, the terminal enzyme of the fatty acid oxidation system. 

CoA, the coenzyme A derivative of acetoacetate, reduces its reactivity as a substrate for /3-ketoacyl-CoA transferase (an enzyme of lipid metabolism) by a factor of 106. Although this requirement for adenosine has not been investigated in detail, it must involve the binding energy between enzyme and substrate (or cofactor) that is used both in catalysis and in stabilizing the initial enzyme-substrate complex (Chapter 6). In the case of /3-ketoacyl-CoA transferase, the nucleotide moiety of coenzyme A appears to be a binding handle that helps to pull the substrate (acetoacetyl-CoA) into the active site. Similar roles may be found for the nucleoside portion of other nucleotide cofactors. 

In the third step, 1, -/3-hydroxyacyl-CoA is dehydrogenated to form /3-ketoacyl-CoA, by the action of /3-hydroxyacyl-CoA dehydrogenase NAD+ is the electron acceptor. This enzyme is absolutely specific for the l stereoisomer of hydroxyacyl-CoA The NADH formed in the reaction donates its electrons to NADH dehydrogenase, an electron carrier of the respiratory chain, and ATP is formed from ADP as the electrons pass to 02. The reaction catalyzed by /3-hydroxyacyl-CoA dehydrogenase is closely analogous to the malate dehydrogenase reaction of the citric acid cycle (p. XXX). 

The fourth and last step of the /3-oxidation cycle is catalyzed by acyl-CoA acetyltransferase, more commonly called thiolase, which promotes reaction of /3-ketoacyl-CoA with a molecule of free coenzyme A to split off the carboxyl-terminal two-carbon fragment of the original fatty acid as acetyl-CoA The other product is the coenzyme A thioester of the fatty acid, now shortened by two carbon atoms (Fig. 17-8a). This reaction is called thiolysis, by analogy with the process of hydrolysis, because the /3-ketoacyl-CoA is cleaved by reaction with the thiol group of coenzyme A... 

CoA-requiring cleavage of the resulting /3-ketoacyl-CoA by thiolase, to form acetyl-CoA and a fatty acyl-CoA shortened by two carbons. The shortened fatty acyl-CoA then reenters the sequence. 

Fig. 2. Metabolic pathways for PHA biosyntheis in fad mutant E. coli strains used in this study. Enoyl-CoA hydratase, epimerase, and 3-ketoacyl-CoA or ACP reductase have been suggested to supply PHA precursors from inhibited b-oxidation pathway. The crosses indicate inactivation of corresponding enzymes. The question mark represents uncharacterized enzyme. Enzymes involved in the metabolic pathways shown have been described previously FabG (21,32), YfcX (24,33), MaoC (34), PhaA (36), and PhaB (36).

Oxidation of 3-hydroxyacyl CoA to 3-ketoacyl CoA producing NADH (catalyzed by hydroxyacyl CoA dehydrogenase). 

Cleavage, or thiolysis, of 3-ketoacyl CoA by a second CoA molecule, giving acetyl CoA and an acyl CoA shortened by two carbon atoms (catalyzed by (3-ketothiolase). 

Palmitate contains 16 carbons, with (14 X 2) + 3 = 31 hydrogens, so each two-carbon unit contains about 4/31 or about 1/8 of the total 3H present. Thus, the counts per minute expected per acetyl-CoA, with two of the four acetyl hydrogens labeled (the other two arising from unlabeled water), is (2/4)(2.48 X 108 cpm//xmol)(l/8) = 1.6 X 107 cpm/jumol, somewhat higher than observed. Exchange between /3-ketoacyl-CoA and solvent water could cause loss of 3H. 

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