title: Identification of the catalytic glutamate in the E1 component of human pyruvate dehydrogenase.
abstract: The pyruvate dehydrogenase complex catalyzes the conversion of pyruvate to acetyl-CoA. The first component (E1) converts pyruvate to bound acetaldehyde using thiamine diphosphate (ThDP) and Mg2+ as cofactors. There is no 3D structure of E1 available but those of other ThDP-dependent enzymes show some similarities including a glutamate residue that assists in ThDP activation. Eukaryotic E1 has an alpha2beta2 structure and the conserved Glu89 of the beta-subunit was identified as a possible catalytic residue by sequence alignment. Human E1 was expressed in Escherichia coli and purified. Mutating Glu89 to glutamine, aspartate and alanine markedly reduces catalytic activity and the affinity for ThDP, consistent with a role as the catalytic glutamate.
authors.affiliation: Department of Molecular Biochemistry, Jilin University, Changchun, PR China.
authors.forename: R
authors.initials: R
authors.lastname: Fang
authors.forename: P F
authors.initials: PF
authors.lastname: Nixon
authors.forename: R G
authors.initials: RG
authors.lastname: Duggleby
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000235:genetics*; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D030481:Pyruvate Dehydrogenase (Lipoamide); D011768:Pyruvate Dehydrogenase Complex / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D016415:Sequence Alignment; D013835:Thiamine Pyrophosphate / Q000378:metabolism
chemical_list: D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D018698:Glutamic Acid; D030481:Pyruvate Dehydrogenase (Lipoamide); D013835:Thiamine Pyrophosphate