Thiolase activity

The last three steps of this four-step sequence are catalyzed by either of two sets of enzymes, with the enzymes employed depending on the length of the fatty acyl chain. For fatty acyl chains of 12 or more carbons, the reactions are catalyzed by a multienzyme complex associated with the inner mitochondrial membrane, the trifunctional protein (TFP). TFP is a heterooctamer of 4/34 subunits. Each a subunit contains two activities, the enoyl-CoA hydratase and the /3-hydroxyacyl-CoA dehydrogenase the /3 subunits contain the thiolase activity. This tight association of three enzymes may allow efficient substrate channeling from one active site to the... 

Very long chain fatty acids are initially oxidized in the peroxisome where the initial oxidation step is catalyzed by acyl-CoA oxidase and the subsequent steps in fS-oxidation are catalyzed by a multi-enzyme complex with hydratase, dehydo-genase, and thiolase activities. Unsaturated fatty acids require additional enzymatic activities, including enoyl-CoA isomerase and dienoyl-CoA reductase. Readers are directed to Vance and Vance (2) for additional details regarding fi-oxidation, including the details of the metabolic reactions. 

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,... 

The medium-chain 3-ketoacyl-CoA thiolase and the 3-ketoacyl-CoA thiolase activity of the trifimctional protein are inhibited by acetyl-CoA. " We have also demonstrated the sensitivity of the trifunctional protein to [acetyl-CoA]/[CoA] but at a fixed [acetyl-CoA + CoASHj. This was carried out by incubating isolated trifunctional protein with 2-hexadecenoyl-CoA, NAD, CoASH, carnitine acetyltransferase and a range of concentrations of carnitine and acetyl-carnitine to keep the total [CoA] constant. Results from a typical titration are shown in Fig. 3. 

Hence, were disposal of acetyl-CoA to keh enesis, the tricarhoxylic acid cycle or to acetyl-carnitine inhibited, feedback inhibition of P-oxidation would result and it has been suggested that this may control p-oxidation in cardiac mitochondria. However, although 3-ketoacyl-CoA esters are readily observed as intermediates of peroxisomal P-oxidation, we have never observed accumulation of 3-ketoacyl-CoA esters in mitochondrial incubations even at maximal P-oxidation flux and under conditions in which acetyl-CoA accumulates. Therefore, the accumulation of 3-ketoacyl-CoA esters may be strongly prevented and an excess of thiolase activity puBs P-oxidation as the... 

After the activation to its coenzyme A ester of acetoacetate by succinyl-CoA 3-oxoacid transferase, the acetoacetyl-coenzyme A must be cleaved into acetyl-CoA by thiolase. There are three thiolase enzymes one cytosolic enzyme and two mitochondrial enzymes (distinguished by the laboratory property of one being activated by potassium and the other is not). The main enzyme only cleaves acetoacetyl-CoA and provides a baseline thiolase activity. The other enzyme cleaves both acetoacetyl-CoA and... 

Genes for poly(3HB) biosynthesis have been introduced into Black Mexican sweet corn (maize, Zea mays) and expressed in cultured cells (Hahn et al. 1997). During a 2-year period in a bioreactor, the introduced phbB and phbC genes were unstable, whereas the thiolase activity was constant. Also, the transformed plant cells grew more slowly than the wild-type cells. The construction of suitable plant species for the production of PHAs in specific cellular locations and at the right time will depend on progress in... 

Figure 4 Peroxisomal fatty-acid (FA) /3-oxidation pathways. While saturated long-chain fatty acids (LCFA) are preferentially degrade in mitochondria, saturated very-long-chain fatty acids (VLCFA) and some LCFA are shortened by peroxisomal /3-oxidation. Degradation of pristanic acid, the product of phytanic acid a-oxidation, and the conversion of the cholesterol-derived 27-carbon bile-acid precursors dihydroxycholestanoic acid (DHCA) and trihydroxycholestanoic acid (THCA) to 24-carbon bile acids also require this pathway. The mechanism by which these substrates enter peroxisomes is unknown. Four enzymatic reactions serve to shorten the substrates by either two (LCFA, VLCFA) or three (pristanic acid, DHCA, THCA) carbon atoms. The 2-methyl group of the latter substrates is shown in brackets. SCPx thiolase refers to the thiolase activity of sterol carrier protein x.

A second case was reported briefiy with a slightly different presentation at 7 months of age in a family in which two siblings had died earlier at 6 months. Blood 3-hydroxybutyrate and acetoacetate concentrations were 29 and 1.3 mmol 1 respectively during acidotic periods and less than 0.2 during symptom-free periods. Liver 3-hydroxybutyrate dehydrogenase activity and kidney, brain and cultured fibroblast acetoacetyl-CoA thiolase activities were normal, but succinyl-CoA 3-keto acid-CoA transferase activity was absent from these tissues (Spence et aL, 1973). However, no detailed report has yet appeared to substantiate these observations. 

A second case dscribed as one of acetoacetyl-CoA thiolase deficiency, has been reported by Robinson et al (1979) (Section 10.4.1), in which urinary metabolites included 3-hydroxybutyrate, acetoacetate, 2-methyl-3-hydroxy-butyrate, 2-methylacetoacetate and tiglylglycine. The authors postulated an enzyme deficiency of short-chain mitochondrial 3-keto acid-CoA thiolase, affecting both acetoacetyl-CoA and 2-methylacetoacetyl-CoA metabolism, ascribing a common enzyme to both metabolic pathways. The presentation of this case was very different from that of de Groot et al, (1977), and further study of 2-methylacetoacetyl-CoA thiolase activity by more direct enzyme assays may resolve the question of the distinction of these enzyme systems (see Section 12.3 below also). 

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