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Purification and characterization of human holocarboxylase synthetase
Author(s) -
Ingaramo Maria,
Beckett Dorothy
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.2_supplement.675
Subject(s) - biotin , biochemistry , pyruvate carboxylase , chemistry , isothermal titration calorimetry , cofactor , enzyme , catabolism , adenylate kinase
Human Holocarboxylase Synthetase (HCS) catalyzes transfer of the cofactor biotin to biotin‐dependent carboxylases. Consequently, this enzyme is of fundamental importance for fatty acid synthesis, gluconeogenesis, and amino acid catabolism. In addition, HCS is involved in transcriptional regulation and it has been proposed to biotinylate histones. HCS was purified and subjected to initial biochemical characterization. Recombinant HCS was purified using a new gene fusion expression system that involves tagging with SUMO (Small Ubiquitin‐related MOdifier). Equilibrium sedimentation experiments determined that HCS is a monomer even in the presence of the intermediate biotinoyl‐adenylate. Isothermal titration calorimetry studies indicate that biotin binding is accompanied by a large heat capacity change, and non‐hydrolysable analogs of the biotinoyl‐adenylate intermediate have high affinity for HCS. To study the steady‐state kinetic properties of the HCS‐catalyzed reaction, we developed an assay that detects radioactive biotin incorporation into P67, a biotin‐acceptor fragment from the alpha subunit of the propionyl‐CoA carboxylase. The results set the stage for elucidating HCS specificity at the molecular level, and will ultimately provide insight into the biotin regulatory mechanism of mammalian cells.