Biotin is a water soluble vitamin that is mainly used as a cofactor in carboxylation reactions by a class of enzyme known as biotin-dependent carboxylases. In order to act as a cofactor, the biotin molecule has to be covalently attached to a lysine residue by an enzyme called holocarboxylase synthetase (HCS). Inherited deficiency of the biotin-dependent propionyl-CoA carboxylase (PCC) results in the inborn error of metabolism propionic acidemia. Mutations in either the alpha (PCCA gene) or beta (PCCB gene) subunit of the enzyme have been shown to cause propionic acidemia. Mutation analysis of the PCCB gene have revealed several mutations. However, few PCCalpha mutations have been described. The first goal of this thesis was to determine the molecular etiology of alpha subunit deficiency at the mRNA as well as at the protein level. I found that most mutations destabilized either the mRNA or the protein. Two other mutations were found to affect the biotinylation of PCCalpha, defining residues important for the folding of the domain or for interaction with HCS. The second part of my thesis was to study in more details the interactions between HCS and the biotinylation domain of PCCalpha, represented by the last 67 amino acids of the subunit (p-67). I expressed and purified p-67 from Pichia pastoris. I compared p-67 with the E. coli biotinylation domain (BCCP87) as substrates for the E. coli orthologous enzyme BirA, using steady-state as well as stopped-flow kinetics. I noticed some differences between these two substrates and how it might relate to the biotinylation reaction. I generated N-terminal and C-terminal deletions of HCS and I tested their activity in vivo and in vitro using purified susbtrates. I was able to map the minimal sequence requirement for HCS activity to the last 348 amino acids of the enzyme. I also found that some longer HCS were either almost or totally inactive or some that were active showed a differential activity towards the different susb
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.37572 |
| Date | January 1999 |
| Creators | Campeau, Eric. |
| Contributors | Gravel, Roy (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Biology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001687464, proquestno: NQ55308, Theses scanned by UMI/ProQuest. |
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