Nonenzymatic biotinylation of a biotin carboxyl carrier protein: unusual reactivity of the physiological target lysine

Protein Sci. 2006 Aug;15(8):1928-35. doi: 10.1110/ps.062187306. Epub 2006 Jul 5.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Acetyl-CoA Carboxylase / chemistry*
  • Adenosine Monophosphate / analogs & derivatives*
  • Adenosine Monophosphate / chemistry
  • Amino Acid Sequence
  • Apoproteins / chemistry
  • Biotin / analogs & derivatives*
  • Biotin / chemistry
  • Biotinylation / methods*
  • Carrier Proteins / chemistry*
  • Fatty Acid Synthase, Type II
  • Lysine / chemistry*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization


  • 5'-AMP-biotin
  • Apoproteins
  • Carrier Proteins
  • Adenosine Monophosphate
  • Biotin
  • Fatty Acid Synthase, Type II
  • Acetyl-CoA Carboxylase
  • biotin carboxyl carrier protein
  • Lysine