Site‐directed mutagenesis of a loop at the active site of E1 (α₂β₂) of the pyruvate dehydrogenase complex

Abstract

Limited proteolysis of the pyruvate decarboxylase (E1, alpha2beta2) component of the pyruvate dehydrogenase (PDH) multienzyme complex of Bacillus stearothermophilus has indicated the importance for catalysis of a site (Tyr281-Arg282) in the E1alpha subunit (Chauhan, H.J., Domingo, G.J., Jung, H.-I. & Perham, R.N. (2000) Eur. J. Biochem. 267, 7158-7169). This site appears to be conserved in the alpha-subunit of heterotetrameric E1s and multiple sequence alignments suggest that there are additional conserved amino-acid residues in this region, part of a common pattern with the consensus sequence -YR-H-D-YR-DE-. This region lies about 50 amino acids on the C-terminal side of a 30-residue motif previously recognized as involved in binding thiamin diphosphate (ThDP) in all ThDP-dependent enzymes. The role of individual residues in this set of conserved amino acids in the E1alpha chain was investigated by means of site-directed mutagenesis. We propose that particular residues are involved in: (a) binding the 2-oxo acid substrate, (b) decarboxylation of the 2-oxo acid and reductive acetylation of the tethered lipoyl domain in the PDH complex, (c) an "open-close" mechanism of the active site, and (d) phosphorylation by the E1-specific kinase (in eukaryotic PDH and branched chain 2-oxo acid dehydrogenase complexes).