Site-directed mutagenesis of a loop at the active site of E1 (alpha2beta2) of the pyruvate dehydrogenase complex. A possible common sequence motif.
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).
Fries
Markus
M
Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, Sanger Building, Old Addenbrooke's Site, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
Chauhan
Hitesh J
HJ
Domingo
Gonzalo J
GJ
Jung
Hyo-Il
HI
Perham
Richard N
RN
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D006358:Hot Temperature; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D011768:Pyruvate Dehydrogenase Complex