Phosphopantetheine-reactive

CoA to form malonyl CoA using C02 in the form of bicarbonate HC03 (Fig. 2). This reaction is catalyzed by the enzyme acetyl CoA carboxylase which has biotin as a prosthetic group, a common feature in C02-binding enzymes. One molecule of ATP is hydrolyzed in the reaction, which is irreversible. The elongation steps of fatty acid synthesis all involve intermediates linked to the terminal sulfhydryl group of the phosphopantetheine reactive unit in ACP phosphopantetheine is also the reactive unit in CoA. Therefore, the next steps are the formation of acetyl-ACP and malonyl-ACP by the enzymes acetyl transacylase and malonyl transacylase, respectively (Fig. 2). (For the synthesis of fatty acids with an odd number of carbon atoms the three-carbon propionyl-ACP is the starting point instead of malonyl-ACP.)... 

Attachment of phosphopantetheine to proteins is catalyzed by a phosphotransferase that utilizes CoA as the donor. A phosphodiesterase removes the phosphopantetheine, providing a turnover cycle.15, 5b A variety of synthetic analogs have been made.4 16 The reactive center of CoA and phosphopantetheine is the SH group, which is carried on a flexible arm that consists in part of the (3-alanine portion of pantothenic acid. A mystery is why pantoic acid, a small odd-shaped molecule that the human body cannot make, is so essential for life. The hydroxyl group is a potential reactive site and the two methyl groups may enter into formation of a "trialkyl lock" (p. 485), part of a sophisticated "elbow" or shoulder for the SH-bearing arm. 

Phosphopantetheine, lipoic acid, and biotin, by virtue of their long, flexible structures, facilitate the physical translocation of chemically reactive species among separate catalytic sites. 

The proximity of the -ketoacyl synthase and phosphopantetheine thiols was confirmed in studies using the bifunctional reagent l,3-dibromo-2-propanone, where the two thiols could be effectively cross-linked by the 5 A-long reagent [61,62]. This reagent cross-linked the two reactive thiols in such a way that two a subunits were concomitantly cross-linked. This important finding is the basis for the conclusion that the -ketoacyl synthase/AGP active site is formed from residues derived from two different a subimits. 

Once the hexameric structure of the yeast FAS was established, the number of functional active sites still remained to be determined. Earlier studies had shown that the functional complex contains approximately six equivalents each of two prosthetic groups 4 -phosphopantetheine [60,63], necessary for the AGP functionality, and flavin mononucleotide [64], an essential component of the enoyl reductase activity. These studies provided an early indication that each of the six active sites in the complex has a full set of the chemical groups necessary for fatty acid synthesis. Nevertheless, conflicting reports appeared in the literature as to the competence of six active sites. Whereas some reports suggested the possibility of half-sites reactivity (only three of the six sites are catalytically competent) [65, 66], others proposed that all six active sites could synthesize fatty acids [62]. Subsequent active site titration experiments were performed which quantitated the amount of fatty acyl products formed in the absence of turnover [67]. Single-turnover conditions were achieved through the use of... 

Following loading of acetyl and malonyl groups onto the P subunit of the enzyme, additional intramolecular transfers must occur to prepare the substrates for the decarboxylative condensation reaction which is catalyzed by the -ketoacyl synthase domain of the a subunit. The end result of these transfers is the thio-esterification of malonate by the phosphopantetheine thiol and of acetate by Cys-1305(a) of the -keto synthase active site. This cysteine has been shown to have a dramatically lowered pK (<5), which would encourage its reactivity [65]. 

Although transfer of the acetate group from its inital site, bound as a serine ester, to its reactive position on Cys-1305(a) can occur via an intermediate in which the acetate is attached to the phosphopantetheine thiol, evidence suggests that this reaction is not kinetically competent and that the biologically significant mechanism utilizes a direct transfer from Ser-819( ) to Cys-1305(a) [73]. Transfer of the malonyl group from its initial position at Ser-5421 (/3) to its reactive position as a phosphopantetheine thioester occurs directly [69]. 

Figure 22.21. Phosphopantetheine. Both acyl carrier protein and CoA include phosphopantetheine as their reactive units.

The answer is c. (Murray, pp 627-661. Scriver, pp 3897-3964. Sack, pp 121—138. Wilson, pp 287-320.1 The almost universal carrier of acyl groups is coenzyme A (CoA). However, acyl carrier protein (ACP) also functions as a carrier ol acyl groups. In fatty acid synthesis, ACP carries the acyl intermediates. The reactive prosthetic group of both ACP and CoA is a phosphopantetheine sulfhiydryl. In ACP, the phosphopantetheine group is attached to the 77-residue polypeptide chain via a serine hydroxyl. In CoA, the phosphopantetheine is linked to the 5 -phosphate of adenosine that is phosphorylated in its 3 -hydroxyl. 

The structure of the phosphopantetheine group, the reactive group common to coenzyme A and acyl carrier protein, is highlighted in yellow. 

Fig. 8.12. CoA and biotin, activation-transfer coenzymes. A. Coenzyme A (CoA or CoASH) and phosphopantetheine are synthesized from the vitamin pantothenate (pantothenic acid). The active sulfhydryl group, shown in blue, binds to acyl groups (e.g., acetyl, succinyl, or fatty acyl) to form thioesters. B. Biotin activates and transfers CO2 to compounds in car-boxylation reactions. The reactive N is shown in blue. Biotin is covalently attached to a lysine residue in the carboxylase enzyme.

Thus the biological importance of the phosphopantetheine group as a catalytic centre is widespread. Numerous examples of the role of coenzyme A are known and the list of phosphopantetheine enzyme centres is growing. The principal reactive element is the thiol, although other attributes of the unique peptide will undoubtedly prove important. The thiol serves as the site of thioester formation and its particular chemical attributes facihtate acyl transfer, carbon chain modification and condensation reactions. The phosphopantetheine thiol represents the most... 

Protein-bound phosphopantetheine has been found in recent years to be involved in acyl binding and reaction in much the same manner as coenzyme A A 77 amino acid protein was isolated from E. coli which acted as an acyl carrier in fatty acid synthesis. This protein completely lacked cysteine or other thiol amino acid, yet functioned by binding various acyl intermediates as thioesters. The reactive centre was phosphopantetheine linked to the protein through a phosphodiester bridge to serine. Similar acyl carrier proteins, or ACPs, have now been isolated from a variety of organisms and extensively characterized. An active ACP protein chain has even been prepared synthetically. ACP per se has been... 

Treatment of the enzyme with acyl phosphate in the complete absence of reduced cofactor has allowed the thiol enzyme derivative to be prepared and separated from its reaction mixture. This in turn has permitted considerable characterization of the enzyme thiol. No special cofactor is involved. The thiol of a cysteine residue from the main peptide chain of the enzyme provides the reactive centre. This enzyme demonstrates that the acyl transfer role of thioesters in biological systems is not restricted to phosphopantetheine and dihydrolipoate derivatives. The reactions of the... 

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