Acyl-thioester

A variety of cellular and viral proteins contain fatty acids covalently bound via ester linkages to the side chains of cysteine and sometimes to serine or threonine residues within a polypeptide chain (Figure 9.18). This type of fatty acyl chain linkage has a broader fatty acid specificity than A myristoylation. Myristate, palmitate, stearate, and oleate can all be esterified in this way, with the Cjg and Cjg chain lengths being most commonly found. Proteins anchored to membranes via fatty acyl thioesters include G-protein-coupled receptors, the surface glycoproteins of several viruses, and the transferrin receptor protein. 

This enzyme [EC 5.1.1.11], also known as phenylalanine racemase (ATP-hydrolyzing), catalyzes the reaction of ATP with L-phenylalanine to produce o-phenylalanine, AMP, and pyrophosphate. In this unusual racemase reaction, a thiol group of an enzyme-bound pantotheine forms a thiolester from an initial aminoacyl-AMP intermediate then, as is typical of acyl thioesters, the a-proton becomes labile, thereby permitting reversible inversion of configuration to produce an equilibrated mixture of thiolester-bound enantiomers. Hydrolysis of the thiolester yields the product. 

Grunnet, I. and Knudsen, J. 1978. Medium chain acyl-thioester hydrolase activity in goat and rabbit mammary fatty acid synthetase complexes. Biochem. Biophys. Res. Commun. 80, 745-749. 

Knudsen J., Clark S. and Dils R. (1976) Purification and some properties of a medium-chain acyl-thioester hydrolase from lactating-rabbit mammary gland which terminates chain elongation in fatty acid synthesis. Biochem. J. 160, 683-691. 

Sex pheromones in the Lepidoptera are multi-component mixtures consisting mostly of olefinic compounds possessing a terminal aldehyde, alcohol, or acetate moiety. Besides functional group differences, the constituents of lepidopteran sex pheromones vary in hydrocarbon chain length and in the specific number, location, and geometry of double bonds. These chemical structures are formed in biosynthetic pathways involving a limited number of enzymatic steps believed to use fatty-acyl thioesters of coenzyme A (acyl-CoA) as substrates. Key reactions are desaturation, limited [3-oxidation, and a small number of terminal functional group modifications (reviewed in Chapter 3). 

Step 6 begins with a nucleophilic addition reaction to the aldehyde group of glyceraldehyde 3-phosphate by a thiol group of an enzyme to form a hemithioacetal, which is oxidized by NAD+ to an acyl thioester. Nucleophilic acyl substitution by phosphate yields the product 1,3-bisphosphoglycerate. 

DH Dehydratase, found in fatty acid syntheases and polyketide syntheases, dehydrates the 3-OH of acyl thioester... 

Despite their enormous structural diversity, polyketide metabolites are related by their common derivation from highly functionalised carbon chains whose assemblies are controlled by multifunctional enzyme complexes, the polyketide synthases (PKSs) which, like the closely related fatty acid synthases, catalyse repetitious sequences of decarboxylative condensation reactions between simple acyl thioesters and malonate, as shown in Fig. 3 [7]. Each condensation is followed by a cycle of modifying reactions ketoreduction, dehydration and enoyl reduction. In contrast to fatty acid biosynthesis where the full cycle of essentially reductive modifications normally follow each condensation reduction, the PKSs can use this sequence in a highly selective and controlled manner to assemble polyketide intermediates with an enormous number of permutations of functionality along the chain. As shown in Fig. 3, the reduction sequence can be largely or entirely omitted to produce the classical polyketide intermediate which bears a carbonyl on every alternate carbon and which normally cyclises to aromatic polyketide metabolites. On the other hand, the reductive sequence can be used fully or partially after each condensation to produce highly functionalised intermediates such as the Reduced polyketide in Fig. 3. Basic questions to be answered are (i) what is the actual polyketide intermediate... 

Acyl Halides (CO-X) > Acyl Anhydrides (-CO-O-OCR) > Acyl Thioester (-CO-SR) > Acyl Esters (-CO-OR) > Acyl Amides (-CO-NR2)... 

DEPC in combination with NEtj has proved to be a new efficient reagent for the direct C-formylation of active methylene compounds with carboxylic acids and also for the iV-acylation (peptide bond formation), 5-acylation (thioester formation) and O-acylation (esterification).4,5 Reaction of DEPC with carboxylic acids 11 in the presence of triethylamine produces transient acyl cyanides, which in the presence of alcohols or thiols results in the formation of the corresponding esters (12) or thioesters (13). 

Carbon-carbon bond formation is a reaction of fundamental importance to the cellular metabolism of all living systems and includes alkylation reactions involving one and five carbon fragments as well as carboxylation reactions. In addition, a very common method of generating carbon-carbon bonds in biology includes the reactions of enolates and their equivalents (such as enamines) with aldehydes, ketones, keto acids, and esters. Reactions in which the enolate derives from an acyl thioester are Claisen condensations, whereas the remainder are classified as aldol reactions. 

In the first half of the ping-pong bi-bi reaction, the active site cysteine is acylated by either an acyl-ACP (KAS I and KAS II) or an acetyl-CoA (KAS III), or in the case of the M. tuberculosis KAS III (FabH) a long-chain acyl-CoA. Attack of the active site cysteine thiolate on the donor acyl thioester is aided by the dipole of the active site helix and an oxyanion hole composed of two backbone NH groups. The additional catalytic residues (His, His or His, Asn) are thought to primarily function in the decarboxylation of malonyl-ACP and stabilization of the acetyl-ACP carbanion that is formed. The carbanion subsequently attacks the acyl-enzyme thioester leading to the formation of the (3-ketoacyl-ACP product. 

Fatty acyl-CoA ligases (specific for short, medium, or long chain fatty acids) catalyze formation of fatty acyl thioester conjugate with coenzyme A (Diagram)... 

The export of the acyl moieties to the cytoplasm depends on the activities of both acyl-thioester esterases and acyl-thloester synthetases. Both Andrews and Keegstra (15) and Block eta/(16) have reported that the acylCoA thloesterase Is located In the Inner membrane and the acylCoA synthetase is located in the outer membrane of the chloroplast. It is assumed that the physiological substrate for the thloesterase of the inner membrane Is the ACP derivative. The combination of these two activities achieves the transport of newly synthesized fatty acids to the cytoplasm where, as the CoA derivatives, they are available for the synthesis of phospholipids in the mitochondria and endoplasmic reticulum. 

Cyanobacteria (c) Release acyl-thioesters or transfer final products directly to lipid... 

A polyketide chain is formed by the condensation between an acyl thioester intermediate and an acyl carrier protein-bound malonyl or methylmalonyl thioester previously selected and transferred to the acyl carrier protein (AGP) by the AT (acyl-transferase) domain. The mechanism of this KS (ketosynthase)-catalyzed reaction. 

Oxidation Acyl thioester (C ) Acetyl-CoA (n = even), propionyl-CoA n = odd) Acetyl-CoA (n = even), propionyl-CoA (n = odd) NAD+ flavoprotein... 

In-chain oxygenation Acyl thioester (C ) Various oxygenated fatty acids Various oxygenated fatty acids O2... 

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... 

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