Holocarboxylase synthetase lysine

Biotin functions to transfer carbon dioxide in a small number of carboxylation reactions. A holocarboxylase synthetase acts on a lysine residue of the apoenzymes of acetyl-CoA carboxylase, pymvate carboxylase, propi-onyl-CoA carboxylase, or methylcrotonyl-CoA carboxylase to react with free biotin to form the biocytin residue of the holoenzyme. The reactive intermediate is 1-7V-carboxybiocytin, formed from bicarbonate in an ATP-dependent reaction. The carboxyl group is then transferred to the substrate for carboxylation (Figure 21—1). 

Biotin is bound covalently to enzymes by a peptide fink to the s -amino group of a lysine residue, forrningbiotinyl-s-arnino-lysine orbiocytin (see Figure 11.1). This postsynthetic modification is catalyzed by holocarboxylase synthetase with the intermediate formation of biotinyl-5 -AMP. In bacteria, this intermediate also acts as a potent repressor of all four enzymes of biotin synthesis. 

Holocarboxylase synthetase from a wide variety of species wUl act on all four apocarboxylases from other species and on a variety of bacterial biotin-dependent apoenzymes. In all the biotin-dependent enzymes investigated to date, the reactive lysine residue is flanked by methionine residues on both sides, and there is a high degree of conservation of the amino sequence around this Met-Lys-Met sequence (Chapman-Smith and Cronan, 1999a, 1999b). 

Biotin serves as a covalently bound coenzyme for acetyl-CoA carboxylases (ACC) 1 and 2, pyruvate carboxylase (PC), propionyl-CoA carboxylase (PCQ and 3-methylcrotonyl-CoA carboxylase (MCQ in mammals and other metazoans (Zempleni et al. 2009). Additional carboxylases exist in microbes (Knowles 1989). The attachment of biotin to the s-amino group of a spedlic lysine residue in holocarboxylases is catalysed by holocarboxylase synthetase (HLCS) or microbial orthologs such as BirA. Biotinylation of carboxylases requires ATP to produce the energy-rich intermediate biotinyl-5 -AMP (Zempleni et al. 2009). 

Holocarboxylase synthetase (EC 6.3.4.10) (HCS or biotin ligase) catalyzes biotin incorporation into various carboxylases which are essential for housekeeping cellular metabolism, as for example ACCase that realizes the first committed step in fatty acid biosynthesis. These carboxylases are synthesized at first as inactive apoproteins which are then modified into active holocarboxylases by post-translational addition of D-biotin to a specific Lysine residue in the apoprotein. This covalent reaction occurs via an amide linkage between the biotin carboxyl group and the e-amino group of a unique Lysine residue present in a specific sequence of all biotin-dependent carboxylases. This covalent attachment occurs in two steps (1) and (2) as follows ... 

Holocarboxylase synthetase. This two-step reaction first activates biotin by forming the adenylate and then attaches the biotin moiety to the s-amino group of a lysine on the carboxylase. 

In biotin-dependent enzymes the cofactor is covalently linked to the protein through an amide bound to a lysine residue (Fig. 7). The attachment of free biotin to apo-enzymes is catalyzed by holocarboxylase synthetases (HCS). This reaction occurs in two steps, an activation step yielding biotinyl-5 -AMP (Eq. 1), which then reacts in a second step with the lysine e-amino group of the apoenzyme, yielding the active holoenzyme (Eq. 2) ... 

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