P-Ketoacyl-CoA thiolase

The subsequent steps of p-oxidation are catalysed by enoyl-CoA hydratase, 3-L-hydroxyacyl-CoA dehydrogenase and p-ketoacyl-CoA thiolase, and lead to... 

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. 

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. 

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. 

In the last step of P-oxidation, p-ketoacyl CoA reacts with coenzyme A in the presence of the enzyme, thiolase. The products of this reaction are acetyl CoA and an acyl CoA containing two carbons less than the original acyl CoA molecule that underwent oxidation. 

The bond between the a- and p-carbons of the P-ketoacyl CoA is cleaved by a thiolase that requires coenzyme A Acetyl CoA is produced from the two carbons at the carboxyl end of the original fatty acyl CoA, and the remaining carbons form a fatty acyl CoA that is two carbons shorter than the original fatty acyl CoA. 

The answer is d. (Murray, pp 230-267. Scriver, pp 2297-2326. Sack, pp 121-138. Wilson, pp 287-320.) Fatty acids must be activated before being oxidized. In this process, they are linked to CoA in a reaction catalyzed by thiokinase (also known as acyl CoA synthetase). ATP is hydrolyzed to AMP plus pyrophosphate in this reaction. In contrast, the enzyme thiolase cleaves off acetyl CoA units from p-ketoacyl CoA, while it forms thioesters during P oxidation. 

Fig. 8. P-Oxidation of fatty acids in E. coli. Long-chain fatty acids are transported into the cell by FadL and converted to their CoA thioesters by FadD (not shown). The acyl-CoAs are substrates for the (1) acyl-CoA dehydrogenase (YafH) to form a trans-2-enoyl-CoA. The double bond is reduced by (2) rrans-2-enoyl-hydratase (crotonase) activity of FadB. The P-hydroxyacyl-CoA is then a substrate for the NADP -dependent dehydrogenase activity of FadB (3). A thiolase, FadA (4), releases acetyl-CoA from the P-ketoacyl-CoA to form an acyl-CoA for subsequent cycles. (5) Polyunsaturated fatty acyl-CoAs are reduced by the 2,4-dienoyl-CoA reductase (FadH). (6) FadB also catalyzes the isomerization of c/s-unsaturated fatty acids to trans. (7) The epimerase activity of FadB converts O-P-hydroxy thioesters to their L-enantiomers via the /rans-2-enoyl-CoA.

Fig. 2. Model of the functional and physical organization of P-oxidation enzymes in mitochondria. (A) P-Oxidation system active with long-chain (LC) acyl-CoAs (B) P-oxidation system active with medium-chain (MC) and short-chain (SC) acyl-CoAs. Abbreviations T, camitineiacylcamitine translocase CPT 11, carnitine palmitoyltransferase 11 AD, acyl-CoA dehydrogenase EH, enoyl-CoA hydratase HD, t-3-hydroxyacyl-CoA dehydrogenase KT, 3-ketoacyl-CoA thiolase VLC, very-long-chain.

The last reaction of p-oxidation, the CoA-dependent cleavage of 3-ketoacyl-CoA, is catalyzed by 3-ketoacyl-CoA thiolase. Three 3-ketoacyl-CoA thiolases encoded by different genes have been detected in rat [27]. Thiolase A is constitutively expressed, whereas the expression of thiolase B is highly induced in response to peroxisomal... 

CoA oxidase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and 3-ketoacyl-CoA thiolase. Dietary sesamin also increased the activity of 2,4-dienoyl-CoA reductase and A, A -enoyl-CoA isomerase, enzymes involved in the auxiliary pathway for p-oxidation of unsaturated fatty acids [246], On the other hand, the results obtained by Fukuda et al. [247] suggest that increased fatty acid oxidation by dietary sesamin leads to decreased esterification of fatty acids and reduces the synthesis and secretion of triacylglycerol. 

The enzyme thiolase catalyzes the cleavage of the P-ketoacyl-CoA a molecule of CoA is required for the reaction. The products are acetyl-GoA and an acyl-CoA that is two carbons shorter than the original molecule that entered the p-oxidation cycle. The CoA is needed in this reaction to form the new thioester bond in the smaller acyl-CoA molecule. This smaller molecule then undergoes another round of the p-oxidation cycle. 

The negative charge of glutamate-462 was found to be necessary for increasing the thermostability of the multienzyme complex, and amidation of the T carboxyl group of glutamate-462 is known to have an adverse effect on the 3-ketoacyl-CoA thiolase activity associated with the small subunit of the fatty acid oxidation complex.These findings provided evidence that a Glu Gin mutation of mitochondrial trifunctional P-oxidation complex, which corresponds to the Glu -> Gin mutation described above,... 

Inhibition of the 3-ketoacyl-CoA thiolase step (with the likely accumulation of 3-ketoacyl-CoA esters) or the 3-hydroxyacyl-CoA dehydrogenase, with accumulation of 3-hydroxyacyl-CoA esters, could lead to feedback inhibition of Poxidation (see Fig. 1). The 3-hydroxyacyl-CoA dehydrogenases, enoyl-CoA hydratases and short-medium-and long- chain acyl-CoA dehydrogenases can be all be inhibited by 3-ketoacyl-CoA esters. The enoyl-CoA hydratases catalyse an equilibrium but can be inhibited by their 3-hydroxyacyl-CoA products. Finally, the acyl-CoA dehydrogenases are subject to inhibition by their 2-enoyl-CoA products and, hence, feedback inhibition has been viewed as potentially very important in the regulation and control of P-oxidation flux. 

We conclude that P-oxidation flux can be controlled by the [NAD ]/[NADH] and [acyl-CoA]/[CoA] ratios in intact mitochondrion. The gross intramitochondrial [NAD ]/[NADH] ratio may not exert control directly over P-oxidation because of the channelling of NAD(H) between 3-hydroxyacyl-CoA dehydrogenases and complex I. Although control of P-oxidation, by CoA acylation or acetylation and feedback inhibition via the 3-ketoacyl-CoA thiolases, is possible it appears to be unlikely to have much impact in intact mitochondrion because (i) 3-ketoacyl-CoA esters are not observed as intermediates of mitochondrial P-oxidation (ii) the K for CoASH of the 3-ketoacyl-CoA thiolases is comparable to that of mitochondrial CoASH-dependent dehydrogenases and much lower than that of CPT II. However, further work characterising the dependence of mitochondrial P-oxidation on these conserved-moiety cycles is necessary. 

Watmough, N.J., D.M. Turnbull, H.S.A. Sherratt K. Bartlett (1989) 5zoc/zem. J. 262,261-269. Measurement of the acyl-CoA intermediates of p-oxidation by h.p.l.c. with photodiode array detection. Reichmann, H. D.C. DeVivo (1991) Comp. Biochem. Physiol. 98B, 327-331. Coordinate enzymatic activity of beta-oxidation and purine nucleotide cycle in a diversity of muscle and other organs of rat. Yang, S.Y., X.Y. He H. Schulz J. Biol Chem. 262, 13027-13031 Fatty acid oxidation in rat brain is limited by the low activity of 3-ketoacyl-CoA thiolase. 

Fig. 2 Metabolic routes for mcl-PHA biosynthesis. Pseudomonas putida GPol synthesizes PHA through P-oxidation and P. putida KT2440 synthesizes PHA through fatty add de novo synthesis. Special PHA consisting of 4-hydroxyalkanoate, 5- hydroxyalkanoate, or 6-hydroxyalkanoate can be produced by various bacteria when suitable precursors are supplied. 1 acyl-CoA synthetase, 2 acyl-CoA dehydrogenase, 3 enoyl-CoA hydratase, 4 NAD-dependent (5)-3-hydroxyacyl-CoA dehydrogenase, 5 3-ketoacyl-CoA thiolase, 6 (ItFspecific enoyl-CoA hydratase, 7 NADPH-dependent 3-ketoacyl-CoA reducatase, 8 3-hydroxyacyl-CoA epimerase, 9 mcl-PHA polymerase, 10 acetyl-CoA carboxylase, 11 malonyl-CoA-acyl carrier protein (ACP) tiansacylase, 12 3-keto-ACP synthase, 13 3-keto-ACP reductase, 14 3-hydroxyacyl-ACP dehydratase, 15 enoyl-ACP reductase, 16 acyl-ACP thiolase, 17 (l )-3-hydroxyacyl-ACP-CoA transacylase, 18 mcl-PHA polymerase...

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