Probing the Mechanism of Inactivation of Human Pyruvate Dehydrogenase by Phosphorylation of Three Sites

• Published in: The Journal of biological chemistry 2001-02, Vol.276 (8), p.5731-5738
• Main Authors: Korotchkina, Lioubov G., Patel, Mulchand S.
• Format: Article
• Language: English
• Subjects: Catalysis; Gene Expression Regulation, Enzymologic; Humans; Mutagenesis, Site-Directed; Mutation; Phosphorylation; Protein Structure, Tertiary; Pyruvate Dehydrogenase Complex - genetics; Pyruvate Dehydrogenase Complex - metabolism; Recombinant Proteins - metabolism; Serine - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M007558200

Activity of the mammalian pyruvate dehydrogenase complex (PDC) is regulated by phosphorylation-dephosphorylation of three serine residues (designated site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) in the α subunit of the pyruvate dehydrogenase (E1) component. Substitutions of the phosphorylation sites were generated by site-directed mutagenesis. Glutamate (S1E) and aspartate (S1D) substitutions at site 1 resulted in the complete loss of PDC activity; however, these mutants were variably active in the decarboxylation and 2,6-dichlorophenolindophenol assays. S1Q had only 3% of wild-type PDC activity. The apparentKm values for pyruvate increased for the mutants of site 1 when determined in the 2,6-dichlorophenolindophenol assay. The substitutions at sites 2 and 3 caused only moderate reductions in activity in the three assays. S3E had a 27-fold increase in the apparent Km for thiamine pyrophosphate and 8-fold increase in the Ki for pyrophosphate. Site 3 was almost completely protected from phosphorylation by thiamine pyrophosphate. The results show that the size rather than negative charge of the substituted amino acid residue affects the active site of E1 and that modification of each of the three serine residues affect the active site in a site-specific manner for its ability to bind the cofactor and substrates.