Differential Effects of Two Mutations at Arginine-234 in the α Subunit of Human Pyruvate Dehydrogenase

The most common mutation in the α subunit of the pyruvate dehydrogenase (E1) component of the human pyruvate dehydrogenase complex (PDC) is arginine-234 to glycine and glutamine in 12 and 3 patients, respectively. Interestingly, these two mutations at the same amino acid position cause E1 (and hence PDC) deficiency by apparently different mechanisms. Recombinant human R234Q E1 had similar Vmax (25.7 ± 4.4 units/mg E1) and apparent Km (101 ± 4 nM) values for TPP as recombinant wild-type human E1, while R234G E1 had no significant change in Vmax (33.6 ± 4.7 units/mg E1) but had a 7-fold increase in its apparent Km value for TPP (497 ± 25 nM). Both of the R234 mutant proteins had similar apparent Km values for pyruvate. Both R234Q and R234G mutant proteins displayed similar phosphorylation rates of sites 1 and 2 by pyruvate dehydrogenase kinase 2 (PDK2) and site 3 by PDK1 compared to wild-type E1. Phosphorylated R234Q E1, R234G E1, and wild-type E1 also had similar dephosphorylation rates of sites 1 and 2 by phosphopyruvate dehydrogenase phosphatase 1. The rate of dephosphorylation of site 3 was about 50% for R234Q E1 and without a significant change for R234G E1 compared to the wild type. The data indicate that the patients with the R234G E1 mutation are symptomatic due to a decreased ability of this mutant protein to bind TPP, whereas the patients with the R234Q E1 mutation are symptomatic due to a decreased rate of dephosphorylation of site 3, hence keeping the enzyme in a phosphorylated/inactivated form.