Biotin carrier protein

FIGURE 21-1 The acetyl-CoA carboxylase reaction. Acetyl-CoA carboxylase has three functional regions biotin carrier protein (gray) biotin carboxylase, which activates C02 by attaching it to a nitrogen in the biotin ring in an ATP-dependent reaction (see Fig. 16-16) and transcarboxylase, which transfers activated C02 (shaded green) from... 

The answer is b. (Murray, pp 627-661. Scriver, pp 3897-3964. Sack, pp 121-138. Wilson, pp 287-320.) The vitamin biotin is the cofactor required by carboxylating enzymes such as acetyl CoA, pyruvate, and propionyl CoA carboxylases. The fixation of CO2 by these biotin-dependent enzymes occurs in two stages. In the first, bicarbonate ion reacts with adenosine triphosphate (ATP) and the biotin carrier protein moiety of the enzyme in the second, the active CO2 reacts with the substrate—e.g., acetyl CoA. 

In the cytosol, acetyl-GoA is carboxylated, producing malonyl-CoA, a key intermediate in fatty-acid biosynthesis (Figure 21.13). This reaction is catalyzed by the acetyl-CoA carboxylase complex, which consists of three enzymes and requires Mn, biotin, and ATP for activity. We have already seen that enzymes catalyzing reactions that take place in several steps frequently consist of several separate protein molecules, and this enzyme follows that pattern. In this case, acetyl-GoA carboxylase consists of the three proteins biotin carboxylase, the biotin carrier protein, and carboxyl transferase. Biotin carboxylase catalyzes the transfer... 

Step 1 biotin is carboxylated using bicarbonate ion (HCO ") as the source of the carboxyl group. Step 2 the carboxylated biotin is brought into proximity with enzyme-bound acetyl-CoA by a biotin carrier protein. Step 3 the carboxyl group is transferred to acetyl-CoA, forming malonyl-CoA. 

These biochemical transformations occur on a multienzyme complex composed of at least three dissimilar proteins biotin carrier protein (MW = 22,000), biotin carboxylase (MW = 100,000) and biotin transferase (MW = 90,000). Each partial reaction is specifically catalyzed at a separate subsite and the biotin is covalently attached to the carrier protein through an amide linkage to a lysyl a-amino group of the carrier protein (338, 339). In 1971, J. Moss and M. D. Lane, from Johns Hopkins University proposed a model for acetyl-CoA carboxylase of E, coli where the essential role of biotin in catalysis is to transfer the fixed CO2, or carboxyl, back and forth between two subsites. Consequently, reactions catalyzed by a biotin-dependent carboxylase proceed though a carboxylated enzyme complex intermediate in which the covalently bound biotinyl prosthetic poup acts as a mobile carboxyl carrier between remote catalytic sites (Fig. 7.13). 

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. 

Pantothenic acid is present in coenzyme A and acyl carrier protein, which act as carriers for acyl groups in metabolic reactions. Pyridoxine, as pyridoxal phosphate, is the coenzyme for several enzymes of amino acid metabolism, including the aminotransferases, and of glycogen phosphorylase. Biotin is the coenzyme for several carboxylase enzymes. 

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. 

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, which is attached to the carrier protein, is presumably able to move by means of its flexible arm from a site on the carboxylase to a site on the transcarboxylase. 

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... 

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... 

Mammalian pyruvate carboxylase has four identical subunits, and the isolated monomer will catalyze the complete reaction. By contrast, three distinct subunits can be isolated from acetyl CoA carboxylase of Escherichia coli and spinach chloroplasts a biotinyl carrier protein, biotin carboxylase, and carboxyl transferase. 

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. 

Biotin in the diet is largely protein bound, and digestion of these proteins by gastrointestinal enzymes produces biotinyl peptides, which may be further hydrolyzed by intestinal bio-tinidase to release biotin. Avidin, a protein found in raw egg whites, binds biotin tightly and prevents its absorption. The peptide biocytin (e-N-biotinyl lysine) is resistant to hydrolysis by proteolytic enzymes in the intestinal tract but together with biotin is readily absorbed. A biotin carrier, the sodium-dependent multivitamin transporter (SMVT)... 

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