title: Differential effects of two mutations at arginine-234 in the alpha subunit of human pyruvate dehydrogenase.
abstract: The most common mutation in the alpha 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 V(max) (25.7 +/- 4.4 units/mg E1) and apparent K(m) (101 +/- 4 nM) values for TPP as recombinant wild-type human E1, while R234G E1 had no significant change in V(max) (33.6 +/- 4.7 units/mg E1) but had a 7-fold increase in its apparent K(m) value for TPP (497 +/- 25 nM). Both of the R234 mutant proteins had similar apparent K(m) 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.
authors.affiliation: Department of Biochemistry, State University of New York at Buffalo, Buffalo, New York 14214, USA.
authors.forename: S J
authors.initials: SJ
authors.lastname: Jacobia
authors.forename: L G
authors.initials: LG
authors.lastname: Korotchkina
authors.forename: M S
authors.initials: MS
authors.lastname: Patel
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine / Q000235:genetics; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004795:Enzyme Stability / Q000502:physiology; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases; D021122:Protein Subunits; D030481:Pyruvate Dehydrogenase (Lipoamide) / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D015325:Pyruvate Dehydrogenase Complex Deficiency Disease / Q000235:genetics*; D013696:Temperature
chemical_list: D007527:Isoenzymes; C000631429:PDK1 protein, human; C000631430:PDK2 protein, human; D021122:Protein Subunits; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D001120:Arginine; D030481:Pyruvate Dehydrogenase (Lipoamide); C065640:pyruvate dehydrogenase E1alpha subunit; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D010744:Phosphoric Monoester Hydrolases