3-Keto thiolase

In the synthesis route from acetyl-CoA to poly(3HB), at least three steps and three enzymes are involved (Fig. 1). The first step is catalyzed by the 3-keto-thiolase (EC 2.3.1.9) which reversibly links two acetyl-CoA moieties to aceto-acetyl-CoA in a Claisen-condensation. The conversion of acetoacetyl-CoA into D-(-)-3-hydroxybutyryl-CoA can be mediated by a reductase (step 2) or via a sequence catalyzed by a reductase (step 4) and two hydratases (steps 5,6). The last step, i.e., the polymerization, is catalyzed by a polymerase (step 3). This... 

If the D-(-)-3-hydroxybutyryl moiety or D-(-)-3-hydroxyvaleryl moiety are derived from butyric acid or valeric acid via acetoacetyl-CoA [28,29], a 3-keto-thiolase is obviously not necessary. 

STEP 4 Chain cleavage. Acetyl CoA is split off from the acyl chain in the final step of jS-oxidation, leaving behind an acyl CoA that is two carbon atoms shorter than the original. The reaction is catalyzed by the enzyme j8-keto thiolase and is mechanistically the reverse of a Claisen condensation reaction (Section 23.8). In the forward direction, a Claisen condensation Joins two estens together to form a j8-keto ester product. In the reverse direction, a retro-Claisen reaction splits a jS-keto ester (or jS-keto thiol ester) apart to form two ester-s (or two thiol esters). 

The major process for activating ketone bodies occurs in the mitochondrion, catalyzed by succinyl CoA-acetoacetate thiotransferase, which, starting with succinyl CoA + acetoacetate, forms succinate + acetoacetyl CoA the latter, via beta keto thiolase, can then react with CoA to produce two acetyl CoAs (Fig. 14.4). Although the GTP that would have been obtained from... 

Ceding, V. and Schlegel, H. G. (1973). b-keto-thiolase from Hydrogenomonas eutropha H16 and its significance in the regulation of poly-b-hydroxybutyrate metabolism. Biochemical Journal 134, 239-248. 

FIGURE 25.12 Elongation of fatty acids in mitochondria is initiated by the thiolase reaction. The /3-ketoacyl intermediate thus formed undergoes the same three reactions (in reverse order) that are the basis of /3-oxidation of fatty acids. Reduction of the /3-keto group is followed by dehydration to form a double bond. Reduction of the double bond yields a fatty acyl-CoA that is elongated by two carbons. Note that the reducing coenzyme for the second step is NADH, whereas the reductant for the fourth step is NADPH. 

A bifnnctional enol-CoA hydratase and 3-hydroxyacetyl-CoA dehydrogenase are used in the degradation of CoA-alkenoic esters to the p-keto acid. This is then degraded to acetyl-CoA and the lower alkanoate ester by 3-ketoacetyl CoA thiolase and acetyl-CoA thiolase. 

Diabetes - insulin dependent Methyl malonic, propionic or isovaleric acidaemias Pyruvate carboxylase and multiple carboxylase deficiency Gluconeogenesis enzyme deficiency glucose-6-phosphatase, fructose-1,6-diphosphatase or abnormality of glycogen synthesis (glycogen synthase) Ketolysis defects Succinyl coenzyme A 3-keto acid transferase ACAC coenzyme A thiolase... 

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. 

The reverse of the above reaction is a key step in the oxidative degradation of fatty acids. This reverse Claisen condensation (catalyzed by thiolase) involves the cleavage of a carbon-carbon bond of a /3-keto ester of coenzyme A by another molecule of coenzyme A to give a new acyl derivative (RCO—SCoA) and ethanoyl (acetyl) derivative (CH3CO—SCoA) ... 

Certain CoA thioester using enzymes catalyze reactions at the fS-carbon or other carbons of the acyl group more distant from the thioester functionality. The fatty acid fi-oxidation cycle provides some examples (Fig. 3). Fatty acids 7 enter the cycle by initial conversion to the CoA ester 8, which is then oxidized to the a,P-unsaturated thioester 9 by a flavin-dependent enzyme. Addition of water to the double bond to form the fi-hydroxy thioester 10 is catalyzed by the enzyme crotonase, which is the centerpiece of the crotonase superfamily of enzymes that catalyze related reactions (37), which is followed by oxidation of the alcohol to form the fi-keto thioester 11. A retro-Claisen reaction catalyzed by thiolase forms acetyl-CoA 12 along with a new acyl-CoA 13 having a carbon chain two carbons shorter than in the initial or previous cycle. 

D) Thiolase removes one carbon from the P-keto intermediate... 

A. During (3-oxidation, a double bond is formed between the a and (3 carbons of a fatty acyl CoA, and FAD is reduced to FADH2. Then water adds across the double bond, and a (3-hydroxy compound is formed. The hydroxyl group on carbon 3 (the 3-carbon) is oxidized to a keto group by NAD+, which is converted to NADH + H+. Finally, a cleavage catalyzed by thiolase releases one acetyl CoA (which contains two carbons). 

Thiolase is an enzyme of the fatty acid oxidation cycle that adds a Co ASH to / -keto-acyl-CoA to convert it to an acyl-CoA with two less carbons plus acetyl-CoA ... 

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