Two human holocarboxylase synthetase proteins display similar kinetic properties

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, in agreement with its role in transcription regulation, it has been proposed that HCS biotinylates histones. There are two major forms of HCS present in human cells that differ by 58 amino acids at the N‐terminus. In order to determine if these residues have any effect on HCS catalytic activity, both forms were recombinantly expressed, purified, and subjected to biochemical characterization. The oligomeric state and kinetic behavior of full length HCS and 58‐HCS were characterized. Equilibrium sedimentation experiments indicate that both HCS forms are monomers even in the presence of the intermediate biotinoyl‐5′‐adenylate. The steady state kinetic analysis as a function of biotin, ATP or biotin accepting substrate concentrations indicate that full length HCS and 58HCS behave similarly. Therefore the additional N‐terminal amino acids do not affect catalysis and are not involved in biotin acceptor fragment recognition. Furthermore, the results indicate that the distinct roles of HCS in transcription regulation and metabolism reflect properties other than the fundamental kinetic characteristics of the two enzyme forms.