Formation of Nε-(biotinyl)lysine in biotin enzymes

Abstract

The role of biotin in carboxyl transfer reactions has been the subject of considerable investigation for a number of years and the mechanisms of the enzymes that catalyze these reactions are known in some detail. The biotin is covalently attached to the enzyme through an amide bond that links the carboxyl group of the valeric acid side chain of biotin to the ɛ-amino group of a lysine residue in the enzyme, yielding a biocytin residue. Although the mechanisms of catalysis of biotin-dependent enzymes have much in common, their structures show considerable diversity. The enzymes vary from structures having the donor site, acceptor site, and biotin attachment site on a single polypeptide chain, as in pyruvate carboxylase of animals, to ones that have each of these three functional sites on separate polypeptides, as in transcarboxylase of Propionibacterium shermanii or acetyl-CoA carboxylase of Escherichia coli . The most common structural form for biotin enzymes appears to be between these two extremes, with the biotin and carboxylation site on one polypeptide and the acceptor site apparently located on a second polypeptide. The covalent attachment of biotin to a specific lysine(s) in apoenzymes is catalyzed by holoenzyme synthetase. The chapter discusses the purification and properties of holocarboxylase synthetase. There are two general methods that have been used to determine the activity of holocarboxylase synthetase. The first procedure follows the incorporation of radioactive biotin into the appropriate apoenzyme directly and the second procedure monitors the appearance of the resultant active holoenzyme.