Carboxyl Carrier

In animals, acetyl-CoA carboxylase (ACC) is a filamentous polymer (4 to 8 X 10 D) composed of 230-kD protomers. Each of these subunits contains the biotin carboxyl carrier moiety, biotin carboxylase, and transcarboxylase activities, as well as allosteric regulatory sites. Animal ACC is thus a multifunctional protein. The polymeric form is active, but the 230-kD protomers are inactive. The activity of ACC is thus dependent upon the position of the equilibrium between these two forms ... 

Bicarbonate as a source of CO2 is required in the initial reaction for the carboxylation of acetyl-CoA to mal-onyl-CoA in the presence of ATP and acetyl-CoA carboxylase. Acetyl-CoA carboxylase has a requirement for the vitamin biotin (Figure 21-1). The enzyme is a multienzyme protein containing a variable number of identical subunits, each containing biotin, biotin carboxylase, biotin carboxyl carrier protein, and transcarboxylase, as well as a regulatory allosteric site. The reaction takes place in two steps (1) carboxylation of biotin involving ATP and (2) transfer of the carboxyl to acetyl-CoA to form malonyl-CoA. 

Protein biotinylation is catalyzed by biotin protein ligase (BPL). In the active site of the enzyme, biotin is activated at the expense of ATP to form AMP-biotin the activated biotin can then react with a nucleophile on the targeted protein. BPL transfers the biotin to a special lysine on biotin carboxyl carrier protein (BCCP), a subunit of AcCoA carboxylase (Scheme 21). Biotinylation of BCCP is very important in fatty acid biosynthesis, starting the growth of the fatty acid with AcCoA carboxylase to generate malonyl-CoA. Recently the crystal structures of mutated BPL and BCCP have been solved together with biotin and ATP to get a better idea of how the transfer fiinctions. ... 

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. 

Biotin 515, 516,711, 721, 723 - 730,723s biosynthesis of 718, 745 in enzymes, table 724 mechanism of action 725 - 729 nutritional requirement 756 Biotin-binding proteins 728 Biotin carboxylase 724 Biotin carboxyl carrier protein 724 Biotin holoenzyme synthetase 724... 

Biochemically, biotin serves as a prosthetic group and a carboxyl carrier for a number of enzymes (179,180). In humans, biotin is required by four carboxylase enzymes ... 

The coenzymatic function of biotin appears to be to mediate the carboxylation of substrates by accepting the ATP-activated carboxyl group and transferring it to the carboxyl acceptor substrate. There is good reason to believe that the enzymatic sites of ATP-dependent carboxylation of biotin are physically separated from the sites at which N -carboxybiotin transfers the carboxyl group to acceptor substrates, that is, the transcarboxylase sites. In fact, in the case of the acetyl-CoA carboxylase from E. coli (see chapter 18), these two sites reside on two different subunits, while the biotinyl group is bonded to a third, a small subunit designated biotin carboxyl carrier protein. 

Structure of A -carboxybiotin linked to biotin carboxyl carrier protein (BCCP). BCCP is one of the components of acetyl-CoA carboxylase isolated from E. coli. 

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. 

Key Words Protein array biotinylation proteomics functional analysis surface capture p53 biotin carboxyl carrier protein fusion protein microarray DNA binding. 

Fig. 1. Crystal structure of the biotin carboxyl carrier protein domain of Escherichia coli. The N- and C-termini and, 50A away, the single lysine residue that is biotinylated in vivo can all clearly be seen. Figure prepared from PDB file 1BDO using Swiss PDB Viewer (19).

Choi-Rhee, E. and Cronan, J. E. (2003) The biotin carboxylase-biotin carboxyl carrier protein complex of Escherichia coli acetyl-CoA carboxylase. J. Biol. Chem. 278, 30,806-30,812... 

The important function of biotin is its role as coenzyme for carboxylase, which catalyses carbon dioxide fixation or carboxylation reaction. The epsilon amino group of lysine in carboxylase enzymes combines with the carboxyl group of biotin to form covalently linked biotinyl carboxyl carrier protein (BCCP or biocytin) (Figure 6.8). This serves as an intermediate carrier of carbon dioxide. The carboxylation of acetyl CoA to malonyl CoA in presence of acetyl CoA carboxylase requires biotin as coenzyme. Propionyl carboxylase and pyruvate carboxylase are also associated with biotin. 

The coenzyme form of pantothenic acid is coenzyme A and is represented as CoASH. The thiol group acts as a carrier of acyl group. It is an important coenzyme involved in fatty acid oxidation, pyruvate oxidation and is also biosynthesis of terpenes. The epsilon amino group of lysine in carboxylase enzymes combines with the carboxyl carrier protein (BCCP or biocytin) and serve as an intermediate carrier of C02. Acetyl CoA pyruvate and propionyl carboxylayse require the participation of BCCP. The coenzyme form of folic acid is tetrahydro folic acid. It is associated with one carbon metabolism. The oxidised and reduced forms of lipoic acid function as coenzyme in pyruvate and a-ketoglutarate dehydrogenase complexes. The 5-deoxy adenosyl and methyl cobalamins function as coenzyme forms of vitamin B12. Methyl cobalamin is involved in the conversion of homocysteine to methionine. 

Acetyl-CoA carboxylase is a biotin-dependent enzyme. It has been purified from microorganisms, yeast, plants, and animals. In animal cells, it exists as an inactive pro-tomer (M.W. 400,000) and as an active polymer (M.W. 4-8 million). The protomer contains the activity of biotin carboxylase, biotin carboxyl carrier protein (BCCP), transcarboxylase, and a regulatory allosteric site. Each protomer contains a biotinyl group bound in amide linkage to the e-amino group of a lysyl residue. 

Mechanism of carboxylation of acetyl-CoA. BCCP = Biotin carboxyl carrier protein Ad = adenosine. 

Numerous studies performed with E. coli have established that, in E. coli, biotin regulates very efficiently its biosynthetic pathway, with an absolute specificity, the biotin vitamers being inactive. As the topics has been largely reviewed, it will be only summarized here. The regulation occurs at the transcriptional level and the biotin operon repressor (BirA) has been well characterized. This 33.5 kDa bifiinctional protein is both an enzyme and a transcriptional regulator (Figure 21). It activates biotin into biotinyl-5 -AMP with ATP (reaction a) and transfers biotin on a specific lysine residue of the biotin accepting proteins (in E. coli, the biotin carboxyl carrier protein (BCCP), a subunit of acetyl-CoA carboxylase) (reaction b). When all the... 

Fig. 1. The acetyl-CoA carboxylase reaction is performed in two steps. Biotin, covalently attached to BCCP (biotin carboxyl carrier protein, accB), is carboxylated by the carboxylase subunit (accC). The heterodimeric transcarboxylase (accA and accD) then transfers the COj to acetyl-CoA, forming malonyl-CoA.

Each ACC half-reaction is catalyzed by a different protein sub-complex. The vitamin biotin is covalently coupled through an amide bond to a lysine residue on biotin carboxyl carrier protein (BCCP, a homodimer of 16.7-kDa monomers encoded by accB) by a specific enzyme, biotin-apoprotein ligase (encoded by birA), and is essential to activity. The crystal and solution structures of the biotinyl domain of BCCP have been determined, and reveal a unique thumb required for activity (J. Cronan, 2001). Carboxylation of biotin is catalyzed by biotin carboxylase (encoded by accC), a homodimeric enzyme composed of 55-kDa subunits that is copurified complexed with BCCP. The accB and accC genes form an operon. The three-dimensional structure of the biotin carboxylase subunit has been solved by X-ray diffraction revealing an ATP-grasp motif for nucleotide binding. The mechanism of biotin carboxylation involves the reaction of ATP and CO2 to form the shortlived carboxyphosphate, which then interacts with biotin on BCCP for CO2 transfer to the I -nitrogen. 

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

Fig. 1. Reactions catalyzed by acetyl-CoA carboxylase. BCCP, biotin carboxyl carrier protein BC, biotin carboxylase CT, carboxyl transferase.

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.)...

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