Search

Enzyme-catalyzed addition of biotin to proteins is highly specific. In any single organism one or a small number of proteins are biotinylated and only a single lysine on each of these proteins is modified. A detailed understanding of the structural basis for the selective biotinylation process has not yet been elucidated. Recently certain mutants of the Escherichia coli biotin protein ligase have been shown to mediate 'promiscuous' biotinylation of proteins. It was suggested that the reaction involved diffusion of a reactive activated biotin intermediate, biotinoyl-5′-AMP, with nonspecific proteins. In this work the reactivity of this chemically synthesized intermediate toward the natural target of enzymatic biotinylation, the biotin carboxyl carrier protein, was investigated. The results indicate that the intermediate does, indeed, react with target protein, albeit at a significantly slower rate than the enzyme-catalyzed process. Surprisingly, analysis of the products of nonenzymatic biotinylation indicates that of five lysine residues in the protein only the physiological target side chain is modified. These results indicate that either the environment of this lysine residue or its intrinsic properties render it highly reactive to nonenzymatic biotinylation mediated by biotinoyl-5′-AMP. This reactivity may be important for its selective biotinylation in vivo. [ABSTRACT FROM AUTHOR]

The binding of the Escherichia coli biotin holoenzyme synthetase to the two ligands, biotin and bio-5′-AMP, is coupled to disorder-to-order transitions in the protein. In the structure of the biotin complex, a 'glycine-rich' loop that is disordered in the apo-enzyme is folded over the ligand. Mutations in three residues in this loop result in significant changes in the affinity of the enzyme for both biotin and bio-5′-AMP. The kinetic basis of these losses in the affinity resides primarily in changes in the unimolecular rates of dissociation of the complexes. In this work, isothermal titration calorimetry has been employed to examine the detailed thermodynamics of binding of three loop mutants to biotin and bio-5′-AMP. The energetic features of dissociation of the protein•ligand complexes also have been probed by measuring the temperature dependencies of the unimolecular dissociation rates. Analysis of the data using the Eyring formalism yielded entropic and enthalpic contributions to the energetic barrier to dissociation. The thermodynamic results coupled with the known structures of the apo-enzyme and biotin complex have been used to formulate a model for progression from the ground-state complex to the transition state in biotin dissociation. In this model, the transition-state is characterized by both partial disruption of noncovalent bonds and acquisition of some of the disorder that characterizes the glycine-rich loop in the absence of ligand. [ABSTRACT FROM AUTHOR]