RESUMEN
The pyruvate dehydrogenase complex (PDHc) is a key megaenzyme linking glycolysis with the citric acid cycle. In mammalian PDHc, dihydrolipoamide acetyltransferase (E2) and the dihydrolipoamide dehydrogenase-binding protein (E3BP) form a 60-subunit core that associates with the peripheral subunits pyruvate dehydrogenase (E1) and dihydrolipoamide dehydrogenase (E3). The structure and stoichiometry of the fully assembled, mammalian PDHc or its core remained elusive. Here, we demonstrate that the human PDHc core is formed by 48 E2 copies that bind 48 E1 heterotetramers and 12 E3BP copies that bind 12 E3 homodimers. Cryo-electron microscopy, together with native and cross-linking mass spectrometry, confirmed a core model in which 8 E2 homotrimers and 12 E2-E2-E3BP heterotrimers assemble into a pseudoicosahedral particle such that the 12 E3BP molecules form six E3BP-E3BP intertrimer interfaces distributed tetrahedrally within the 60-subunit core. The even distribution of E3 subunits in the peripheral shell of PDHc guarantees maximum enzymatic activity of the megaenzyme.
Asunto(s)
Microscopía por Crioelectrón , Complejo Piruvato Deshidrogenasa , Humanos , Complejo Piruvato Deshidrogenasa/metabolismo , Complejo Piruvato Deshidrogenasa/química , Modelos Moleculares , Dihidrolipoamida Deshidrogenasa/metabolismo , Dihidrolipoamida Deshidrogenasa/química , Multimerización de Proteína , Unión Proteica , Subunidades de Proteína/metabolismo , Subunidades de Proteína/química , Acetiltransferasa de Residuos Dihidrolipoil-Lisina/metabolismo , Acetiltransferasa de Residuos Dihidrolipoil-Lisina/químicaRESUMEN
The Escherichia coli pyruvate dehydrogenase complex (PDHc) is a ~5 MDa assembly of the catalytic subunits pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). The PDHc core is a cubic complex of eight E2 homotrimers. Homodimers of the peripheral subunits E1 and E3 associate with the core by binding to the peripheral subunit binding domain (PSBD) of E2. Previous reports indicated that 12 E1 dimers and 6 E3 dimers bind to the 24-meric E2 core. Using an assembly arrested E2 homotrimer (E23), we show that two of the three PSBDs in the E23 dimerize, that each PSBD dimer cooperatively binds two E1 dimers, and that E3 dimers only bind to the unpaired PSBD in E23. This mechanism is preserved in wild-type PDHc, with an E1 dimer:E2 monomer:E3 dimer stoichiometry of 16:24:8. The conserved PSBD dimer interface indicates that PSBD dimerization is the previously unrecognized architectural determinant of gammaproteobacterial PDHc megacomplexes.
Asunto(s)
Dihidrolipoamida Deshidrogenasa , Acetiltransferasa de Residuos Dihidrolipoil-Lisina , Escherichia coli , Complejo Piruvato Deshidrogenasa , Dihidrolipoamida Deshidrogenasa/genética , Dihidrolipoamida Deshidrogenasa/química , Dihidrolipoamida Deshidrogenasa/metabolismo , Acetiltransferasa de Residuos Dihidrolipoil-Lisina/química , Acetiltransferasa de Residuos Dihidrolipoil-Lisina/metabolismo , Dimerización , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Complejo Piruvato Deshidrogenasa/química , Complejo Piruvato Deshidrogenasa/metabolismoRESUMEN
This review summarizes the recent developments on the regulation of human pyruvate dehydrogenase complex (PDC) by site-specific phosphorylation by four kinases. Mutagenic analysis of the three phosphorylation sites of human pyruvate dehydrogenase (E1) showed the site-independent mechanism of phosphorylation as well as site-independent dephosphorylation of the three phosphorylation sites and the importance of each phosphorylation site for the inactivation of E1. Both the negative charge and size of the group introduced at site 1 were involved in human E1 inactivation. Mechanism of inactivation of E1 was suggested to be site-specific. Phosphorylation of site 1 affected E1 interaction with the lipoyl domain of dihydrolipoamide acetyltransferase, whereas phosphorylation site 3 appeared to be closer to the thiamine pyrophosphate (TPP)-binding region affecting coenzyme interaction with human E1. Four isoenzymes of pyruvate dehydrogenase kinase (PDK) showed different specificity for the three phosphorylation sites of E1. All four PDKs phosphorylated sites 1 and 2 in PDC with different rates, and only PDK1 phosphorylated site 3. PDK2 was maximally stimulated by the reduction/acetylation of the lipoyl groups of E2. Presence of the multiple phosphorylation sites and isoenzymes of PDK is important for the tissue-specific regulation of PDC under different physiological conditions.