Pathogenic mutations in the carboxyl-terminal domain of glutaryl-CoA dehydrogenase: effects on catalytic activity and the stability of the tetramer.

Inherited defects in glutaryl-CoA dehydrogenase cause the neurometabolic disease, glutaric acidemia type I. Five of over 80 mutations that have been identified are located in a carboxyl-terminal domain. The five mutations were generated by site directed mutagenesis and expressed in Escherichia coli. The mutant dehydrogenases were purified and characterized by circular dichroism and fluorescence spectroscopy, analytical size exclusion chromatography, thermal stability, and steady state kinetic analysis. There is no significant change in the alpha-helical content of the mutant proteins and little effect on tertiary structure; however, spectral properties of the mutant proteins indicate that the FAD prosthetic group can dissociate from the mutant proteins. Size exclusion chromatography shows that four mutant proteins dissociate to dimers or a mixture of monomers and dimers. Steady state kinetic analyses show that K(m) for glutaryl-CoA is affected by the mutations, but there is little effect on k(cat) compared with the wild type dehydrogenase. The lack of effects of the mutations on the K(m) for the electron acceptor, electron transfer flavoprotein, and on secondary structure suggests that the mutations do not result in long-range structural effects. The crystal structures of the acyl-CoA dehydrogenases show that their overall folding patterns are very similar and that the carboxyl-terminal domain is involved in substrate binding, FAD binding and intersubunit interactions. Investigations of mutations in the carboxyl-terminal domain of glutaryl-CoA dehydrogenase clearly illustrate these multiple roles of this domain. The results also indicate that a primary effect of the mutations is to cause alterations that promote aggregation.