Human Holocarboxylase Synthetase (HCS) transfers the vitamin biotin to the biotin carboxyl carrier protein (BCCP) domains of five biotin-dependent carboxylases. There are two major forms of HCS present in mammalian cells, which differ by 57 amino acids at their N-terminus. Both variants catalyze biotinylation through a two step reaction in which an activated intermediate, bio-5'-AMP, is first synthesized from biotin and ATP. In the second step, the biotin moiety is covalently attached to a specific lysine residue on the carboxylase. The mammalian carboxylases comprise the cytosolic acetyl-CoA carboxylase 1 (ACC1), the outer mitochondrial membrane bound acetyl-CoA carboxylase 2 (ACC2), and the three mitochondrial carboxylases pyruvate carboxylase (PC), 3-methylcrotonyl-CoA carboxylase (MCC) and propionyl-CoA carboxylase (PCC).

In order to investigate the HCS reaction mechanism and specificity, both isoforms were recombinantly expressed, purified, and biochemically characterized. The basic mechanistic features of the two HCS variants were investigated using steady state and pre-steady state kinetic methods. The latter methods allow the determination of the rates of bio-5'-AMP synthesis and biotin transfer independent of each other. Both isoforms catalyze the overall reaction similarly and synthesize bio-5'-AMP with a rate of 0.1s-1. Biotin transfer to the BCCP domain fragments of ACC1 and ACC2 carboxylases, to which HCS has continuous access from the cytosol, is slow and saturable. In contrast, biotin transfer to the BCCP domain fragments from the mitochondrial carboxylases PC, PCC and MCC is characterized by rates that are significantly faster and limited by the collision of enzyme and acceptor substrate. The same pre-steady state methods were applied to two biotin ligases from archaea and prokarya, and showed that this collision limited biotin transfer mechanism is widespread among evolutionary domains.

The observation of a collision limited reaction emphasizes the role of protein substrate recognition in the biotinylation reaction, and provides a mechanism for establishing a hierarchy among carboxylases that favors mitochondrial substrates. This default hierarchy can be overridden according to cellular demands by modifying the carboxylases' expression. The results support the idea that in the cell one of the mechanisms of biotin-related metabolism regulation relies on HCS specificity to control biotin distribution among carboxylases.