title: Selectivity in post-translational biotin addition to five human carboxylases.
abstract: Human holocarboxylase synthetase (HCS) catalyzes linkage of the vitamin biotin to the biotin carboxyl carrier protein (BCCP) domain of five biotin-dependent carboxylases. In the two-step reaction, the activated intermediate, bio-5'-AMP, is first synthesized from biotin and ATP, followed by covalent linkage of the biotin moiety to a specific lysine residue of each carboxylase BCCP domain. Selectivity in HCS-catalyzed biotinylation to the carboxylases was investigated in single turnover stopped flow and quench flow measurements of biotin transfer to the minimal biotin acceptor BCCP fragments of the carboxylases. The results demonstrate that biotinylation of the BCCP fragments of the mitochondrial carboxylases propionyl-CoA carboxylase, pyruvate carboxylase, and methylcrotonoyl-CoA carboxylase is fast and limited by the bimolecular association rate of the enzyme with substrate. By contrast, biotinylation of the acetyl-CoA carboxylase 1 and 2 (ACC1 and ACC2) fragments, both of which are accessible to HCS in the cytoplasm, is slow and displays a hyperbolic dependence on substrate concentration. The correlation between HCS accessibility to biotin acceptor substrates and the kinetics of biotinylation suggests that mitochondrial carboxylase sequences evolved to produce fast association rates with HCS in order to ensure biotinylation prior to mitochondrial import. In addition, the results are consistent with a role for HCS specificity in dictating biotin distribution among carboxylases.
authors: Department of Chemistry and Biochemistry, Center for Biological Structure and Organization, University of Maryland, College Park, Maryland 20742, USA.
authors: Maria
authors: M
authors: Ingaramo
authors: Dorothy
authors: D
authors: Beckett
mesh_terms: D000103:Acetyl-CoA Carboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001710:Biotin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019921:Biotinylation / Q000502:physiology*; D019755:Carbon-Carbon Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054889:Fatty Acid Synthase, Type II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D001710:Biotin; D019755:Carbon-Carbon Lyases; D054889:Fatty Acid Synthase, Type II; D019731:Carbon-Nitrogen Ligases; C023312:holocarboxylase synthetases; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein