Carboxyltransferases

Some enzymes are nonfunctional until posttranslationally modified. Examples of these enzymes include the acyl- and carboxyltransferases. While lipoate and phosphopantetheine are necessary for acyl transfer chemistry, tethered biotin is used in carboxyl transfer chemistry. Biotin and lipoate tethering occur under a similar mechanism the natural small molecule is activated with ATP to form biotinyl-AMP or lipoyl-AMP (Scheme 20). A lysine from the target protein then attacks the activated acid and transfers the group to the protein. The phosphopantetheine moiety is transferred using its own enzyme, the phosphopantetheinyltrans-ferase (PPTase). The PPTase uses a nucleophilic hydroxy-containing amino acid, serine, to attach the phosphopantetheinyl (Ppant) arm found in coenzyme A to convert the apo (inactive) carrier protein to its holo (active) form. The reaction is Mg -dependent. 

This enzyme [EC 2.1.3.1], also known as methylmalonyl-CoA carboxyltransferase, catalyzes the reaction of (5 )-2-methyl-3-oxopropanoyl-CoA with pyruvate to produce propanoyl-CoA and oxaloacetate. The enzyme requires biotin, cobalt, and zinc as cofactors. 

The much studied E. coli enzyme is composed of a 156-residue biotin carboxyl carrier protein,38 a 449-residue biotin carboxylase, whose three-dimensional structure in known 39/393 and a carboxyltransferase subunit consisting of 304 (a)- and 319 (P)- residue chains. These all associate as a dimer of the three subunits (eight peptide chains).40-42... 

The biotin carboxyl carrier subunit of E. coli acetyl-CoA carboxylase contains the covalently bound biotin.55a/b The larger biotin carboxylase subunit catalyzes the ATP-dependent attachment of C02 to the biotin and the carboxyltransferase subunit catalyzes the final transcarboxylation step (Eq. 14-5, step b) by which acetyl-CoA is converted into malonyl-CoA. 

Reactions catalyzed by acetyl-CoA carboxylase. In E. coli, BCCP and the two enzymatic activities (biotin carboxylase and carboxyltransferase) can be separated from each other. In contrast, in the liver all three components exist on a single multifunctional polypeptide. 

Acetyl-CoA carboxylase of E. coli is a multienzyme complex that consists of three protein components that can be isolated individually Biotin carboxyl carrier protein (BCCP), biotin carboxylase, and carboxyltransferase (fig. 

Subsequently, the C02 is transferred from BCCP to acetyl-CoA in a reaction catalyzed by carboxyltransferase, which yields malonyl-CoA. 

An active, cobalt-containing, oxaloacetate transcarboxylase (methyl-malonyl-CoA pyruvate carboxyltransferase) has been isolated from Propionobacterium shermanii grown with 60Co2+ (137). The metal content corresponds to two equivalents of 60Co(II) per mole of enzyme. 

One ligand, NCI-65828, was found to inhibit AccD5 (an essential acyl-CoA carboxylase carboxyltransferase domain) competitively with an experimental K of 13.1 pM... 

Animal and fungal ACCs are comprised of large multifunctional polypeptides containing the biotin carboxylase, biotinyl carboxyl carrier protein, and carboxyltransferase... 

Fig. 2. Acetyl-CoA carboxylase. (A) Eukaryotic ACCs contain -2300 residues organized into three functional domains — biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT). The role of the region between the biotin carboxyl carrier and carboxyltransferase domains is unknown. The biotin carboxyl carrier protein contains a typical conserved biotin attachment-site motif, VMKMV. The sites of phosphorylation are indicated by asterisks. (B) Electron micrograph of polymerized rat acetyl-CoA carboxylase (F. Ahmad, 1978). (C) Crystal structure of the biotin carboxylase domain of the yeast enzyme. In the presence of soraphen A, the biotin carboxyl carrier protein domain forms an inactive monomer the likely position of the modeled ATP-binding site is shown (adapted from Ref. [2]). (D) Crystal structure of the dimeric carboxyltransferase domain of the yeast enzyme. Although acetyl-CoA was included in the crystallization, density was observed only for CoA at one site and adenine at the other (adapted from Ref. [2]). (E) NMR structure of the biotin carboxyl carrier apoprotein domain of the human ACC2 The lysine attachment site for biotin is shown (RIKEN Structural Genomics/Proteomics Initiative, 2006). (See color plate section, plate no. 3.)...

The only multisite Ping-Pong mechanism known in 1970 was that of transcarboxylase (methylmalonyl-CoA carboxyltransferase) (33), but a number have been identified since then, including not only reactions in which biotin, lipoic acid, and 4-phosphopantetheine are carriers between active sites, but also reactions where oxidation and reduction of a group on the enzyme occur at different sites [e.g., glutamate synthase (34)]. 

In prokaryotes and in plastids of some plants, the ACC is a multisubunit enzyme, whereas in eukaryotes the cytosolic isozyme and, in some instances also the plastid isozyme, are multidomain proteins. The latter contain three major functional domains, which account for the biotin carboxylase (BT), biotin carboxyl-carrier (BCC) and carboxyltransferase (CT) activities and, which are organized in one large polypeptide. 

The multisubunits enzyme is encoded by the nuclear DNA, with the exception of the j8-subunit of carboxyltransferase that is encoded by a chloroplastic gene [11]. In grasses, the chloroplastic multidomain ACC is encoded by a nuclear gene, which is distinct from that coding for the cytosolic multidomain ACC. 

Phosphorylation of serine residue(s) of the j8-subunit of the carboxyltransferase unit occurs in pea chloroplasts incubated in the light [20]. Alkaline phosphatase treatment reduces ACC activity in parallel to removal of phosphate groups from ACC. This activation by phosphorylation is opposite to the inhibition of animal ACC by phosphorylation but is consistent with the increase in ATP concentration and rates of fatty acid synthesis in chloroplasts in the light and the activation of other plastid enzymes by phosphorylation. These results suggest that the CT subunit reaction is rate determining for overall ACC activity, at least for the multisubunits enzyme of dicots. 

Widely used commercial herbiddes, represented by AOPPs and CHDs, are potent inhibitors of ACCs of sensitive plants and kill them by shutting down fatty acid biosynthesis, thus leading to metabolite leakage from the membranes and cell death [27]. AOPPs and CHDs inhibit the carboxyltransferase activity (Scheme 9.1, reachon [2]), thus blocking the transfer of the carboxyl group to acetyl-CoA [28]. They show nearly competitive inhibition with respect to the substrate acetyl coenzyme A [29]. 

This reaction, which proceeds in two half-reactions, a biotin carboxylase (BC) reaction and a carboxyltransferase (CT) reaction is the first committed step in fatty acid biosynthesis and is the rate limiting reaction for the pathway [103]. 

Figure 5. Acetyl-coenzyme-A carboxylase (ACC) has critical roles in fatty acid metabolism. The ACC-catalyzed biotin carboxylase (BC) and carboxyltransferase (CT) reactions [103].

De novo synthesis of fatty acids requires the combined action of acetyl-CoA carboxylase and fatty acid synthetase. The acetyl-CoA carboxylases contain biotin carboxyl carrier protein (BCCP), biotin carboxylase ahd carboxyltransferase. The reaction proceeds in two steps. Firstly, the biotin moiety of BCCP is carboxylated. Secondly, the carboxyl group is transferred to the acceptor acetyl-CoA. This latter reaction proceeds in a concerted fashion (Mildvan et aL, 1966). The mechanism of the carboxylation/decarboxylation reaction has been recently probed using the model compound N-1 -methoxy carbonylbiotin methyl... 

In contrast, the carboxylase from Escherichia coli readily dissociates into its constituent proteins, BCCP, biotin carboxylase and carboxyltransferase. BCCP is a dimer of apparently identical subunits of mol.wt. 22 500. Biotin carboxylase is a dimer of identical units of mol.wt. 51 000. The carboxyltransferase is an a2)82-tetramer with 30000 and 35000 subunits (cf. Volpe and Vagelos, 1976). Neither citrate nor phosphorylation play any role in regulating the bacterial enzyme. Instead guanosine nucleotides, guanosine 3 -diphosphate 5-di-(and tri-) phosphate are used. These inhibit the carboxyltransferase. 

Acetyl-CoA carboxylase has been purified from several plant tissues. In wheat germ, the BCCP and biotin carboxylase are associated with one fraction while the carboxyltransferase can be isolated independently (Heinstein and Stumpf, 1969). The carboxylase from chloroplasts has prokaryotic ... 

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