Thioester Thiolase

The final step in the /3-oxidation cycle is the cleavage of the /3-ketoacyI-CoA. This reaction, catalyzed by thiolase (also known as j8-ketothiolase), involves the attack of a cysteine thiolate from the enzyme on the /3-carbonyI carbon, followed by cleavage to give the etiolate of acetyl-CoA and an enzyme-thioester intermediate (Figure 24.17). Subsequent attack by the thiol group of a second CoA and departure of the cysteine thiolate yields a new (shorter) acyl-CoA. If the reaction in Figure 24.17 is read in reverse, it is easy to see that it is a Claisen condensation—an attack of the etiolate anion of acetyl-CoA on a thioester. Despite the formation of a second thioester, this reaction has a very favorable A).q, and it drives the three previous reactions of /3-oxidation. 

FIGURE 24.17 The mechanism of the thiolase reaction. Attack by an enzyme cysteine thiolate group at the /3-carbonyl carbon produces a tetrahedral intermediate, which decomposes with departure of acetyl-CoA, leaving an enzyme thioester intermediate. Attack by the thiol group of a second CoA yields a new (shortened) acyl-CoA. 

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 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... 

Acetyl-CoA is formed from CoA and acetate by the enzyme acetyl-CoA synthetase, an ADP-forming ligase. Phosphotrans-acetylase forms acetyl-CoA from CoA and acetyl-phosphate, which in turn is formed from acetate and ATP catalyzed by acetate kinase. Other enzymes that can form acetyl-CoA from CoA and other acetyl group donors include ATP citrate lyase and thiolase. Longer chain acyl-CoA thioesters are typically formed from CoA and a fatty acid catalyzed by ligases generally known as acyl-CoA synthetases. 

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. 

Vogel KW, Drueckhammer DG. A reversed thioester analogue of acetyl-coenzyme A An inhibitor of thiolase and a synthon for other acyl-CoA analogues. J. Am. Chem. Soc. 1998 120 3275-3283. 

Acetoacetate is formed from acetyl CoA in three steps (Figure 22.19). Two molecules of acetyl CoA condense to form acetoacetyl CoA. This reaction, which is catalyzed hy thiolase, is the reverse of the thiolysis step in the oxidation of fatty acids. Acetoacetyl CoA then reacts with acetyl CoA and water to give 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) and CoA. This condensation resembles the one catalyzed by citrate synthase (Section 17.13). This reaction, which has a favorable equilibrium owing to the hydrolysis of a thioester linkage, compensates for the unfavorable equilibrium in the formation of acetoacetyl CoA. 3-Hydroxy-3-methylglutaryl CoA is then cleaved to acetyl CoA and acetoacetate. The sum of these reactions is... 

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. 

AcetoacetylCoA thiolase (E.C. 2.3.1.9), acetoacetylCoA reductase (E.C. 1.1.1.36), and polyhydroxybutyrate synthetase12471 are the enzymes involved in polyester synthesis. AcetoacetylCoA thiolase catalyzes the head-to-tail Claisen condensation of two acetylCoA molecules. In this reaction, the active site cysteine attacks acetylCoA to form a thioester enzyme intermediate, which then reacts with the enolate derived from enzymatic deprotonation of the other acetylCoA. Mechanistic studies have been performed on this enzyme from Zooglea ramigera, which has been cloned and overexpressed12471. It has been established that the thiolase will form acyl enzyme intermediates with a number of acylCoA substrates, but will only accept acetylCoA as the nucleophile. After subsequent reduction, this results in all polymer units possessing a P-hydroxy group. These polymers are also useful sources of (R)-P-hydroxy acids[2481. 

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.

As we have seen in the section on thiolases, Claisen condensations normally involve activation of the electrophilic carhonyl through formation of a thioester and stabilization of the attacking carbanion also as a thioester. Dihydroxynaphthoyl-CoA synthase (MenB) catalyzes an intramolecular Claisen condensation reaction in which only the nucleophilic portion of the molecule has been converted to a thioester. This reaction is a component of the menaquinone biosynthetic pathway, and most studies have focused on the enzyme from M. tuberculosis based on the premise that this pathway may be a valid target for the development of novel compounds that inhibit both replicating and nonreplicating bacteria. ... 

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 final process for coenzyme A thioester synthesis is by the thiolytic cleavage of /8-keto acyl coen me A derivatives. The thiolase reaction is the principal metabolic process for degrading the hydrocarbon chain of fatty acids. 

An example in which reactivity of both the attacking nucleophile and the electrophilic acceptor is dependent on the special character of acyl thioesters is in the condensation of two acetyl coenzyme A units to form acetoacetyl coenzyme A. This is the reverse of the thiolase reaction... 

There is controversy about almost all the items in this list, but they nevertheless merit discussion. Intuitively one could almost take for granted that ATP would be a requirement since the synthetic process involves overcoming an energy barrier to proceed to a lower entropy level. Simple reversal of the mitochondrial oxidation system should not require ATP, and Seubert et al. (1957) found that none was needed for synthesis by his system of isolated enzymes of the /3-oxidation cycle. The equilibrium of the thiolase reaction (/8-ketoacyl-CoA -f- CoA acyl-[minus 2]-CoA + acetyl-CoA) does not favor synthesis. Seubert et al. (1957) utilized an enoyl reductase which irreversibly reduced CoA thioesters of unsaturated acids to saturated... 

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