Palmitoyl-coenzyme A synthetase. Mechanism of reaction

Bar‐Tana, Jacob; Rose, G.; Brandes, Ruth; Shapiro, B.

doi:10.1042/bj1310199

Cited by 35 publications

(21 citation statements)

References 7 publications

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“…After overexpression in E. coli, some of these enzymes could catalyze isotope exchange between 32 PPi and ATP when incubated with jasmonic acid, salicylic acid, and/or IAA. Isotope exchange is indicative of the adenylation half-reaction carried out by all other types of AAE enzymes (Bar-Tana et al, 1973). Interestingly, addition of CoA to the enzyme assays did not cause release of AMP (Staswick et al, 2002), so it is unlikely that these enzymes are acyl-CoA synthetases.…”

Section: Discussionmentioning

confidence: 99%

“…In this report, we define AAEs as those enzymes that first activate their respective carboxylic acid substrates through the pyrophosphorylysis of ATP, forming an enzyme-bound acyl-AMP intermediate called an adenylate. The carbonyl carbon of the adenylate most commonly then undergoes nucleophilic attack by the free electrons of a thiol group from the acyl acceptor, forming the relevant thioester and releasing AMP (Bar-Tana et al, 1973). The identity of the ultimate acyl acceptor may vary considerably.…”

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confidence: 99%

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Arabidopsis Contains a Large Superfamily of Acyl-Activating Enzymes. Phylogenetic and Biochemical Analysis Reveals a New Class of Acyl-Coenzyme A Synthetases

2003

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Acyl-activating enzymes are a diverse group of proteins that catalyze the activation of many different carboxylic acids, primarily through the formation of a thioester bond. This group of enzymes is found in all living organisms and includes the acyl-coenzyme A synthetases, 4-coumarate:coenzyme A ligases, luciferases, and non-ribosomal peptide synthetases. The members of this superfamily share little overall sequence identity, but do contain a 12-amino acid motif common to all enzymes that activate their acid substrates using ATP via an enzyme-bound adenylate intermediate. Arabidopsis possesses an acyl-activating enzyme superfamily containing 63 different genes. In addition to the genes that had been characterized previously, 14 new cDNA clones were isolated as part of this work. The protein sequences were compared phylogenetically and grouped into seven distinct categories. At least four of these categories are plant specific. The tissue-specific expression profiles of some of the genes of unknown function were analyzed and shown to be complex, with a high degree of overlap. Most of the plant-specific genes represent uncharacterized aspects of carboxylic acid metabolism. One such group contains members whose enzymes activate short-and medium-chain fatty acids. Altogether, the results presented here describe the largest acyl-activating enzyme family present in any organism thus far studied at the genomic level and clearly indicate that carboxylic acid activation metabolism in plants is much more complex than previously thought.Carboxylic acid activation plays a vital role in numerous metabolic pathways in all living organisms. Activation of carboxylic acids provides the precursors for pathways that lead to the biosynthesis and/or breakdown of many types of important metabolites, including lipids, amino acids, sugars, and a variety of secondary metabolites. Given the chemical diversity of substrates requiring activation, several types of carboxylic acid activating enzymes have evolved to fulfill this role. Although some of these enzymes couple carboxyl groups to amines or alcohols, most acyl-activating enzymes are acid-thiol ligases (EC 6.2.1). Even within this smaller group, the similarity in the types of products formed is not strictly reflected in the routes by which these enzymes carry out their respective reactions: at least three different catalytic mechanisms are used to create the various thioester products (Groot et al., 1976; Stein et al., 1996;Sánchez et al., 2000). We are particularly interested in the acyl-activating enzymes (AAEs). This group of enzymes has previously been called the acyl adenylate-forming (Conti et al., 1996;Chang et al., 1997) or AMP-binding protein (Fulda et al., 1997) superfamily. In this report, we define AAEs as those enzymes that first activate their respective carboxylic acid substrates through the pyrophosphorylysis of ATP, forming an enzyme-bound acyl-AMP intermediate called an adenylate. The carbonyl carbon of the adenylate most commonly then undergoes nucleophilic attac...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Arabidopsis Contains a Large Superfamily of Acyl-Activating Enzymes. Phylogenetic and Biochemical Analysis Reveals a New Class of Acyl-Coenzyme A Synthetases

2003

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“…The ACL family catalyzes the formation of a fatty acyl-CoA from a fatty acid substrate, ATP, and CoA in a Mg 2+ -dependent two-step reaction (Bar-Tana et al, 1973a, 1973bBlack et al, 1997) (Fig. 3).…”

Section: Enzyme Kinetics Of the Al Domainmentioning

confidence: 99%

Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase

Fritzler

Zhu

2007

International Journal for Parasitology

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The apicomplexan Cryptosporidium parvum possesses a unique 1,500-kDa polyketide synthase (CpPKS1) comprised of 29 enzymes for synthesizing a yet undetermined polyketide. This study focuses on the biochemical characterization of the 845-amino acid loading unit containing acyl-[ACP] ligase (AL) and acyl carrier protein (ACP). The CpPKS1-AL domain has a substrate preference for long chain fatty acids, particularly for the C20:0 arachidic acid. When using [ 3 H] palmitic acid and CoA as co-substrates, the AL domain displayed allosteric kinetics towards palmitic acid (Hill coefficient, h = 1.46, K 50 = 0.751 μM, V max = 2.236 μmol mg −1 min −1 ) and CoA (h = 0.704, K 50 = 5.627 μM, V max = 0.557 μmol mg −1 min −1 ), and biphasic kinetics towards adenosine 5′-triphosphate (K m1 = 3.149 μM, V max1 = 373.3 nmol mg −1 min −1 , K m2 = 121.0 μM, and V max2 = 563.7 nmol mg −1 min −1 ). The AL domain is Mg 2+ -dependent and its activity could be inhibited by triacsin C (IC 50 = 6.64 μM). Furthermore, the ACP domain within the loading unit could be activated by the C. parvum surfactin production element-type phosphopantetheinyl transferase. After attachment of the fatty acid substrate to the AL domain for conversion into the fatty-acyl intermediate, the AL domain is able to transfer palmitic acid to the activated holo-ACP in vitro. These observations ultimately validate the function of the CpPKS1-AL-ACP unit, and make it possible to further dissect the function of this megasynthase using recombinant proteins in a stepwise procedure.

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“…that reactions (2) and (3) are not part reactions of (1). This possibility W~B tested by presenting intact microsomes, which contain both types of Table 3) that upon saturation with ATP and palmitic acid, the rate of reaction could not be further promoted by the addition of palmitoyl-AMP.…”

Section: Xeparation Of Fractionsmentioning

confidence: 99%

“…When the enzyme was solubilized and purified [2], it showed very high activity in reaction (1) but was almost devoid of activity for reaction (2) and (3) [3]. The latter two reactions are generally considered to be part reactions of palmitoyl-CoA synthesis according to reaction (1).…”

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confidence: 99%

Two Forms of Microsomal Palmitoyl‐Coenzyme A Synthetase

Brandes

Zohar

Arad

et al. 1973

European Journal of Biochemistry

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Two palmitoyl-CoA synthetase activities have been demonstrated in rat liver microsomes. One, catalyzing palmitoyl-CoA synthesis from ATP, palmitate and CoASH as well as from palmitoyl-AMP and CoASH. A second enzyme reacts with the fist constituents only and is inactive with palmitoyl-AMP. The first enzyme, but not the second catalyzes the reaction between palmitoyl-AMP and PPi to yield ATP and palmitic acid.This duality has been shown by: (a) partial separation of the two types of activity by fractional solution in Triton X-100, (b) kinetic behaviour testing competition between both reaction mixtures at saturating concentrations and (c) the varying distribution of the two activities in microsomes obtained from rats under different dietary regimes or when diabetes was induced by alJoxan. When the enzyme was solubilized and purified [2], it showed very high activity in reaction (1) but was almost devoid of activity for reaction (2) and (3) [3]. The latter two reactions are generally considered to be part reactions of palmitoyl-CoA synthesis according to reaction (1). Their disappearance upon purification raised a number of possibilities: (a) That the loss of the part reactions was due to change in the enzyme protein during extraction and purification, making it non-accessible to palmitoyl-AMP. (b) That two enzymes coexist in liver microsomes ; one, which had been purified, catalyzes reaction (I), but not (2) and (3), while a second enzyme catalyzes all three reactions.Indications for the second possibility have been obtained in the experiments, to be presented, involving preparative separation of the two types of activities, mutual competition between the substra- +AMP (2)Enzyme. Palmitoyl-CoA synthetase, acid: CoA ligase-(AMP) (EC 6.2.1.3).tes and the changing distribution of these activities in intact microsomes obtained from livers of rats under differing conditions. and 100-4OOpg microsomal protein in a total volume of 0.6 ml. EDTA was added in order to avoid non-enzymatic CoASH disappearance which otherwise occurred in the presence of ATP. Incubation was carried on for 10 min a t 30 "C and the reaction was stopped by the addition of 0.2 ml 15O/, trichloroacetic MATERIALS AND METHODS Microsomes

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Palmitoyl-coenzyme A synthetase. Mechanism of reaction

Cited by 35 publications

References 7 publications

Arabidopsis Contains a Large Superfamily of Acyl-Activating Enzymes. Phylogenetic and Biochemical Analysis Reveals a New Class of Acyl-Coenzyme A Synthetases

Arabidopsis Contains a Large Superfamily of Acyl-Activating Enzymes. Phylogenetic and Biochemical Analysis Reveals a New Class of Acyl-Coenzyme A Synthetases

Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase

Two Forms of Microsomal Palmitoyl‐Coenzyme A Synthetase

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