Reactivity of Human Holocarboxylase Synthetase Toward Biotin-Accepting Substrates

Abstract

Human Holocarboxylase Synthetase (HCS) catalyzes biotin transfer to carboxylases in a two-step reaction in which the activated biotin, bio-5′-AMP, is first formed from substrates biotin and ATP. The biotin is then transferred to a single lysine residue on the carboxylase. The five substrates for HCS, including acetyl-CoA carboxylases 1 and 2, pyruvate carboxylase, 3-methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase, play critical roles in metabolism. Two isoforms of HCS differ at the amino-terminus by 57 amino acids, and their specificity towards the substrates is unknown. The biochemistry of HCS function has been investigated by characterizing the basic properties of the two isoforms and their interactions with the five acceptor-protein substrates. Equilibrium sedimentation indicates that the proteins are monomers in their apo-forms and when bound to the adenylated intermediate. Steady state analysis of the overall reaction indicates that both isoforms possess similar behavior with respect to the small molecule substrates. In contrast, stopped flow fluorescence measurements of biotin transfer indicate that the HCS forms can exhibit distinct association rates with a single biotin acceptor. Moreover, the isoforms display preferential reactivity among the substrates. These results are consistent with a role for HCS N-terminus in acceptor substrate recognition, and suggest a role for HCS in dictating a hierarchy of biotin utilization by carboxylases.