Propionic acidemia is a rare autosomal recessive disorder of intermediary metabolism. It is caused by a deficiency of the mitochondrial enzyme propionyl-CoA carboxylase (PCC, EC 22.214.171.124), a heteropolymeric protein composed of two subunits, alpha and beta. PCC requires ATP and biotin as cofactors for the reaction, the latter enzymatically added onto the alpha subunit. We investigated coding sequence mutations in the alpha subunit of PCC by analyzing fibroblast RNA from propionic acidemia patients deficient in alpha subunit function by single-strand conformation polymorphism and direct sequencing. Five missense mutations and one short in-frame deletion were found among different patients. Four mutations were located in the putative biotin carboxylase domain, whereas the two others were within the 67-amino-acid C-terminal domain previously shown to be required to obtain biotinylation of the alpha subunit. We analyzed fibroblast extracts for the presence of a biotinylated alpha subunit by Western blot analysis using streptavidin coupled to alkaline phosphatase. Four of five cell lines failed to show a biotinylated alpha subunit, regardless of the position of the mutations within the coding sequence. Two mutations located in the biotinylation domain were expressed in an Escherichia coli-based system and shown to abolish biotinylation of the domain. The results suggest that most mutations have a severe impact on the stability or the functionality of the alpha subunit.
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