4’-Phosphopantetheine residue

Although fatty acid 8-oxidation is catalyzed by a series of intramitochon-drial enzymes, and the fatty acyl chain is carried by CoA, fatty acid synthesis is catalyzed by a cytosolic-multienzyme complex in which the growing fatty acyl chain is bound by thioester linkage to an enzyme-bound 4 -phosphopantetheine residue. This component of the fatty acid synthetase complex is ACP. 

Fig. 52. Schematical representation of fatty acid synthase. B shows the 4-phosphopantetheine residue of the acyl-carrier (AGP) in detail...

This enzyme contains a phosphopantetheine residue, derived from the vitamin pantothenic acid, and a cysteine residue both can form thioesters with acyl groups. The growing fatty acyl chain moves from one to the other of these sulfhydryl residues as it is elongated. 

In bacteria, ACP is a small protein of 77 residues that transports an acyl group from enzyme to enzyme. In vertebrates, however, ACP appears to be a long arm on a multienzyme synthase complex, whose apparent function is to shepherd an acyl group from site to site within the complex. As in acetyl CoA, the acyl group in acetyl ACP is linked by a thioester bond to the sulfur atom of phosphopantetheine. The phosphopantetheine is in turn linked to ACP through the side-chain -OH group of a serine residue in the enzyme. 

Figure 21-2. Fatty acid synthase multienzyme complex. The complex is a dimer of two identical polypeptide monomers, 1 and 2, each consisting of seven enzyme activities and the acyl carrier protein (ACP). (Cys— SH, cysteine thiol.) The— SH of the 4 -phosphopantetheine of one monomer is in close proximity to the— SH of the cysteine residue of the ketoacyl synthase of the other monomer, suggesting a "head-to-tail" arrangement of the two monomers. Though each monomer contains all the partial activities of the reaction sequence, the actual functional unit consists of one-half of one monomer interacting with the complementary half of the other. Thus, two acyl chains are produced simultaneously. The sequence of the enzymes in each monomer is based on Wakil.

FIGURE 21-4 Acyl carrier protein (ACP). The prosthetic group is 4 -phosphopantetheine, which is covalently attached to the hydroxyl group of a Ser residue in ACP. Phosphopantetheine contains the B vitamin pantothenic acid, also found in the coenzyme A molecule. Its —SH group is the site of entry of malonyl groups during fatty acid synthesis. 

In higher animals as well as in My cobacterium,207 yeast,208 and Euglena, the fatty acid synthase consists of only one or two multifunctional proteins. The synthase from animal tissues has seven catalytic activities in a single 263-kDa 2500-residue protein 209 The protein consists of a series of domains that contain the various catalytic activities needed for the entire synthetic sequence. One domain contains an ACP-like site with a bound 4 -phosphopantetheine as well as a cysteine side chain in the second acylation site. This synthase produces free fatty acids, principally the C16 palmitate. The final step is cleavage of the acyl-CoA by a thioesterase, one of the seven enzymatic activities of the synthase. See Chapter 21 for further discussion. 

Apo-ACP is activated by a transferase, holo-ACP synthetase, which transfers 4 -phosphopantetheine from CoA to the hydroxyl group of a serine residue in the apoprotein, releasing ADP. ACP is inactivated by a hydrolase that releases 4 -phosphopantetheine, which can be reutilized for CoA synthesis. 

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. 

MSAS from P. patulum was separated from the FAS via sucrose gradient centrifugation [121,122] and thus shown to constitute a distinct multifunctional enzymatic system. It was purified to homogeneity and found to be a 190 kDa multifunctional enzyme [22,120]. The enzyme was more stable in the presence of its substrates and at mildly basic pH values. The pH optimum of the enzyme was 7.6 and apparent K values for its substrates were 10 pM (acetyl-CoA), 7 pM (malonyl CoA), and 12 pM (NADPH) [115,120,123]. The rate for triacetate lactone formation in the absence of NADPH was determined to be ten-fold lower than for 6-MSA formation (Fig. 5) [120]. Analogous to FASs and peptide synthetases, 4 -phosphopantetheine is a covalently bound cofactor of 6-MSAS [124]. Likewise, iodoacetamide and N-ethylmaleimide were found to inactivate the enzyme, suggesting the presence of catalytic sulfhydryl residues in 6-MSAS [124]. Furthermore, in the presence of malonyl CoA and NADPH, low concentrations of iodoacetamide convert 6-MSAS into a malonyl CoA decarboxylase. Without external addition of acetyl-CoA, 6-MSAS decarboxylates the malonyl group and the derived acetyl moiety is used as a starter unit for the formation of 6-MSA [125]. 

Organisms of all biological kingdoms convert 64 into the cys-teamine derivative phosphopantetheine (65) using L-cysteine as substrate. 65 is converted to coenzyme A (66) by attachment of an adenosine moiety via a pyrophosphate linker and phosphorylation of the ribose moiety. Phosphopantetheine can be attached covalently to serine residues of acyl carrier proteins that are parts of fatty acid synthases and polyketide synthases. 

Our next goal was to establish conditions that would permit acylation of the phosphopantetheinyl thiol with acetate, malonate and fiuther more advanced polyketide intermediates. The reasons for this are twofold. First, we wanted to look for evidence of specific binding of these acyl substituents consistent with the proposed role of the ACP in binding and stabilisation. Secondly, we wished to use these acyl ACPs in in vitro mechanistic studies of the assembly process. As is evident from the stracture of the act ACP, there is an exposed cysteine residue at position 17, and preliminary studies showed, as expected, that selective chemical derivatisation of the phosphopantetheine thiol in the presence of cysteine 17 was not possible. This was overcome by mutagenesis to replace cysteine 17 with a serine, and the C17S mutant has been used in many of our subsequent in vitro studies. 

The cofactor of fatty acid synthase is bound to the enzyme at a point near a specific residue of cysteine. This cysteine residue is important in the catalytic mechanism. T he sulfhydryl group of this cysteine is used for temporarily holding the fatty acid moiety each time a new molecule of maIonic acid is transferred to the 4-phosphopantetheine group. One might refer to the diagram of the enzyme in Chapter 5, where the sulfhydryl groups of the cysteine residue and of 4-phosphopantetheine are shown. 

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. 

Adding up the two reactions, one finds that the aldehyde is oxidized to the corresponding acid, or that the a-kcto-acid is oxidatively decarboxylated to the corresponding acid and CO2, while ATP is assembled, with the thiol playing a catalytic role. This thiol may be a cysteine residue in the enzyme protein or an enzyme-linked phosphopantetheine molecule or coenzyme A, a soluble coenzyme in which phos-phopantetheine is linked to 3 -phospho-AMP or lipoic acid, also known as thioctic acid, a molecule that can serve at the same time as electron carrier and as group carrier, a unique distinction. 

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