Pantetheine cofactors

The catalytic mechanisms and molecular recognition properties of peptide synthetases have been studied for several decades [169]. Nonribosomal peptides are assembled on a polyenzyme-protein template, first postulated by Lipmann [170]. The polyenzyme model was refined into the thiotemplate mechanism (Fig. 11) in which the amino acid substrates are covalently bound via thioester linkages to active site sulfhydryls of the enzyme and condensed via a processive mechanism involving a 4 -phosphopantetheine carrier [171-173].The presence of a covalently attached pantetheine cofactor was first established in a cell-free system that catalyzed enzymatic synthesis of the decapeptides gramicidin S and tyrocidine. As in the case of fatty acid synthesis, its role in binding and translocating the intermediate peptides was analyzed [174,175]. 

The concentration of free pantothenic acid in the liver is about 15 xM that in the heart is about tenfold greater (Robishaw and Neely, 1985). The concentration of the cofactor form of the vitamin, coenzyme A, is higher in the mitochondrion than in the cytosol. In the Ever, cytosolic coenzyme A is about 0.06 mM, and mitochondrial coenzjmie A, about 2.6 mM. In the liver, about 70% of coenzyme A is mitochondrial, whereas in the heart about 95% is mitochondrial (Tahiliani and Neely, 1987). These values might be compared with that for carnitine, another molecule used in the handling of fatty acids. Please consult the Carnitine section in Chapter Four. About half of the coenzjrme A in liver occurs as the long-chain fatty acyl-coenzyme A derivative. The concentration of fatty acid s)mthase in the cytoplasm is quite low, about 0.01 pM. Hence, the concentration of the 4 -phospho-pantetheine cofactor is much lower than that of coenzyme A. The pantothenic acid boimd to this enzyme does not make a significant contribution to our dietary vitamin. 

Figure I Schematic views of reactions involved in peptide biosynthesis. (1) Adenylate formation involving nucleophilic attack of the carboxyl group at the a-phosphate of the MgATP --complex with release of MgPP. - (2) aminoacylation of the pantetheine cofactor by formation of the thio-late anion, attack of the mixed anhydride, and release of AMP (3) tentative view of the peptide bond formation by nucleophilic attack of the aminoacyl-nitrogen at the preceding thioester-car-boxyl, involving deprotonaiion-protonation (4) epimerization of an aminoacyl-thioester, a reaction differing from those catalyzed by the well-characterized amino acid racemases. (Altered from Ref. 13. )...

Low-molecular-mass thiols such as coenzyme A and protein-bound thiol cofactors such as phospho-pantetheine are present in all cells. Their SH groups can also be oxidized to disulfides and it is of interest that in resting bacterial spores these compounds exist largely as disulfides or mixed disulfides. Upon germination of the spores special enzymes reduce the disulfides.136 Some proteins involved in control of protein synthesis contain SH groups that add covalently to C-6 atoms of a uracil ring in specific mRNA molecules. Control of their state of reduction may also be important.137... 

An additional series of reactions,350 which are shown in Eq. 24-38, leads to pantoic acid, pantetheine, coenzyme A, and related cofactors.350a i The initial reactions of the sequence do not occur in the animal body, explaining our need for pantothenic acid as a vitamin. 

One of the reasons for its widespread use is based on the fact that CoA - and the CoA-derived phospho-pantetheine tether found on various carrier proteins - acts as the major carrier of metabolites containing carboxylic acid groups short and long carbon chain acids, Krebs cycle metabolites, and amino acids are the most common compounds carried. However, at any given moment most CoA molecules are involved in the transfer of acetyl groups among a variety of both small and macromolecules, and consequently it is from this role that the cofactor derives the name that Lipmann originally bestowed on it Coenzyme A, to indicate its involvement in activation of acetate. ... 

Characterization of the activities of the horse and pig enzymes has shown that pantetheinase is specific for the pantothenate moiety of its substrate and will not react with CoA, 4 -phosphopantetheine, or /i-alanylcys-teamine. However, it accepts the modification of the cysteamine moiety and hydrolyzes a variety of pantetheine thioesters. The enzyme does not require any cofactors, although it is inhibited by pantetheine (pantetheine disulfide), oxidized glutathione, and thiol inhibitors (such as Hg ), suggesting the involvement of an active site cysteine in catalysis. The pig enzyme has a molecular weight of 72 kDa and exhibits a A n, of 20pmoll for pantetheine. All these studies have been performed on purified or partially purified native enzymes no pantetheinase enzyme has been cloned to date. 

Aminoacylation assays have revealed that overexpressed peptide synthetases are only incompletely pusttranslationally modified by pantetheine. Tyrocidine synthetase 1 expressed in E. coJi contains about 1.5% holo-enzyme (80). The reduced cofactitr content leads to decreased activities in aminoacylation and epimerization reactions, which require the cofactor (64,80,84)- Some evidence has been obtained that apo-eniymes may slightly differ from holo--en2ymes with respect to catalytic properties (80). Addition of 4 -phos-phopantetheine is thought to be catalyzed by a holo-enzyme synthase utilizing CoA ... 

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