MECHANISMS OF EXPRESSION OF PYRUVATE-DEHYDROGENASE DEFICIENCY CAUSED BY AN E1-ALPHA SUBUNIT MUTATION

Objective: To characterize the biochemical mechanisms of expression of the pyruvate dehydrogenase (PDH) E1 alpha subunit exon 10 R302C misse nse mutation. Background: Mutations in the X-linked E1 alpha subunit g ene are responsible for most cases of PDH deficiency, an important cau se of neurodevelopmental defects and neurodegeneration with primary la ctic acidemia. Although the disease shows extreme allelic heterogeneit y, the R302C mutation has been defined in several unrelated cases. Met hods: Cell lines expressing selectively either the mutant or wild-type E1 alpha alleles against identical genetic backgrounds were generated from the fibroblasts of a female heterozygous for the R302C mutation. Enzyme activity, mRNA, polypeptide expression, and turnover were stud ied in each. Results: The residual PDH activity was below measurable l evels in the cell line (B5) expressing only the mutant allele and norm al in the wild-type polypeptide expressing (A10) cell line, confirming that the R302C mutation alone is sufficient to cause a severe PDH def iciency. The mutant polypeptide was less stable than the wild-type pol ypeptide, but the steady-state level of the mutant E1 alpha protein wa s reduced only two- to threefold. Conclusions: The primary mechanism o f expression of the R302C mutation must; be limitation of catalytic ef ficiency. We speculate that catalysis may be inhibited in the mutant p olypeptide because conformational changes are induced near serine 300, a residue that is particularly important as a regulatory phosphorylat ion site in the wild-type polypeptide.