A-Amino group of biocytin

Figure 12-3- The structure of d-biotin and /V-carboxybiotin covalently attached to an -amino group of a lysyl residue of a carboxylase. Biocytin is the biotin similarly attached to the e-amino group of a lysine. The dotted line indicates the amide bond linkage.

As shown in Figure 11.1, biotin is a bicydie compound with fused ureido (im-idazolidone) and thiophene rings, and an aliphatic carboxylate side chain. It is bound covalendy to enzymes by the formation of a peptide bond between the carboxyl group of the side chain and the e-amino group of a lysine residue forming biocytin (biotinyl-lysine). 

Biotin is bound covalently to enzymes by a peptide link to the e-amino group of a lysine residue, forming biotinyl-e-amino-lysine or biocytin (see Figure 11.1). This postsynthetic modification is cattdyzed by holocarboxylase synthetase with the intermediate formation of biotinyl-5 -AMP. In bacteria, this intermediate tdso acts as a potent repressor of till four enzymes of biotin synthesis. 

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. 

The studies of Reed and co-workers on the nature of protein-bound lipoic acid and its enzymatic release and reincorporation may be applicable to biotin-containing enzymes. It is pertinent to note that a conjugated form of biotin, biocytin, has been isolated from yeast autolyzate and identified as A -biotinyl-L-lysine (Wright et ah, 19r)2 Peck et al., 1952). Biotin is now known to be the prosthetic group of several carboxylases (see Ochoa and Kaziro, 1961, for a review of these enzymes). Although the nature of the moiety to which biotin is bound has not been established, it seems highly probable that it is the -amino group of a lysine residue. 

Biotin enzymes carboxylases that use biotin as a cofactor. The biotin is bound via an amide bond to the E-amino group of a specific lysine residue in the enzyme protein, i.e. B.e. contain a biotinyllysyl residue. Free (+)-E-V-biotinyl-L-lysine actually occurs in yeast extract, and is known as biocytin. During catalysis, N-atom 1 of the biotin residue is carboxylated in an ATP-dependent reaction ATP + HCOj" + bioti-nyl-enzyme (I) -> ADP + Pj + carboxybiotinyl-en-zyme (II). The carboxyl group is then transferred from (II) to the carboxylase substrate (II) + substrate —> (I) + carboxylated substrate. 

Amide bonds resulting from the condensation of amino groups of a-amino acids with carboxyhc groups of organic acids are found in many other compounds but these are not classified as peptides. Examples are some vitamins such as pantothenic acid, tetrahydrofolic acid (folic acid with two or more molecules of glutamic acid are already peptides) and the active form of biotin (biocytin). 

Biotin contains a condensed ring system consisting of two five-membered heterocycles (formula in Chapt. VI-5). In the conjugate biocytin, the carboxyl group of biotin is linked peptidically with the -amino group of lysine. A similar bond probably holds the vitamin to the protein molecule in the biotin-containing enzymes, which catalyze carboxylations (see Chapt. XII-6). 

Biocytin is e-N-biotinyl-L-lysine, a derivative of D-biotin containing a lysine group coupled at its e-amino side chain to the valeric acid carboxylate. It is a naturally occurring complex of biotin that is typically found in serum and urine, and probably represents breakdown products of recycling biotinylated proteins. The enzyme biotinidase specifically cleaves the lysine residue and releases the biotin component from biocytin (Ebrahim and Dakshinamurti, 1986, 1987). 

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