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

Abstract

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 ␣ 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 apparent K m 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 K m for thiamine pyrophosphate and 8-fold increase in the K i 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.The mammalian pyruvate dehydrogenase complex (PDC) 1 plays an important role in defining the fate of three-carbon compounds derived largely from carbohydrates and to some extent from amino acids. PDC comprises multiple copies of three catalytic enzymes, namely pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3); in addition, the complex also contains a binding protein referred to as E3-binding protein (E3BP), and two regulatory enzymes, namely pyruvate dehydrogenase kinase (PDK) and phosphopyruvate dehydrogenase phosphatase. E1 carries out the decarboxylation of pyruvic acid and the reductive acetylation of lipoyl moieties covalently linked to E2. E2 then catalyzes the transfer of acetyl groups to CoA, forming acetyl-CoA and fully reduced lipoyl moieties. E3 reoxidizes the reduced lipoyl groups of E2 and transfers the electrons to NAD ϩ , forming NADH. Sixty subunits of mammalian E2 and 12 subunits of E3BP form the inner core structure, whereas 20 -30 tetrameric E1 (␣ 2 ␤ 2 ) components bind this core and 6 E3 homodimers bind to the E3BP subunits (1, 2).Regulation of mammalian PDC activity is accomplished in large part by phosphorylation (resulting in inactivation) of the E1 component by a family of pyruvate dehydrogenase kinases (PDK 1-4 isozymes) and dephosphorylation (leading to activation) of phosphorylated E1 by a set of specific phosphatases (phosphopyruvate dehydrogenase phosphatase 1-2 isozymes) (1, 3-6). The ␣ subunit of the E1 component has three phosphorylation sites, named site 1 (Ser-264), site 2 (Ser-271), and site 3 (Ser-203), and phosphorylation of any one of these three sites results in inactivation (7-9). In vivo inactivati...

Regulation of mammalian pyruvate dehydrogenase complex by phosphorylation: complexity of multiple phosphorylation sites and kinases

“…reductive acetylation of lipoyl groups of E2) is affected to a greater extent by phosphorylation than the decarboxylation step. 1Q E1 had only 3% of control activity in PDC-reaction (Korotchkina and Patel, 2001a) indicating the importance of the volume of the substituted group as well as the charge. Substitutions of sites 2 and 3 with glutamate did not result in the same degree of inactivation as the phosphorylation of sites 2 and 3 did ( Figure 4B).…”

“…Site 1 is localized in the substrate channel according to the structure of BCKDH and may take part in positioning of the substrates during the catalytic reaction (Aevarsson et al, 1999). For this reason the replacement of serine even with alanine resulted in the modified affinity for the first substrate, pyruvate (Korotchkina and Patel, 2001a).…”

“…The acidic residues of the lipoyl domain were found to be important Activities were measured by the CO 2 assay (decarboxylation reaction) by the formation of 14 CO 2 from [1-14 C]pyruvate in the absence of electron acceptors. 2A3A E1 was phosphorylated on site 1 only (1P) by PDK2, 1A3A E1 on site 2 only (2P) by PDK2 and 1A2A E1 on site 3 only (3P) by PDK1 in the presence of human E2-E3BP (Korotchkina and Patel, 2001a). Black bars, wild-type and glutamate-substituted mutant E1s (1E, 2E and 3E), and open bars, double mutants of E1 phosphorylated on the remaining phosphorylation site (1P, 2P and 3P) (Korotchkina and Patel, 2001a).…”

“…2A3A E1 was phosphorylated on site 1 only (1P) by PDK2, 1A3A E1 on site 2 only (2P) by PDK2 and 1A2A E1 on site 3 only (3P) by PDK1 in the presence of human E2-E3BP (Korotchkina and Patel, 2001a). Black bars, wild-type and glutamate-substituted mutant E1s (1E, 2E and 3E), and open bars, double mutants of E1 phosphorylated on the remaining phosphorylation site (1P, 2P and 3P) (Korotchkina and Patel, 2001a). *, not detectable.…”

“…In a recent study the mechanism of inactivation of human E1 was investigated using site-specific replacement of the three phosphorylation sites (Korotchkina and Patel, 2001a). Serines of the phosphorylation sites 1 (Ser264), 2 (Ser271) and 3 (Ser203) were replaced with alanine, glutamate and glutamine.…”