tailieunhanh - Báo cáo khoa học: Site-directed mutagenesis of a loop at the active site of E1 (a2b2) of the pyruvate dehydrogenase complex A possible common sequence motif

Limitedproteolysis of the pyruvate decarboxylase (E1,a2b2 ) component of the pyruvate dehydrogenase (PDH) multi-enzyme complexofBacillus stearothermophilushas indicated the importance for catalysis of a site (Tyr281-Arg282) in the E1asubunit (Chauhan, ., Domingo, ., Jung, . & Perham, . (2000)Eur. J. , 7158–7169). This site appears to be conserved in the a-subunit of hetero-tetrameric 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-. . | Eur. J. Biochem. 270 861-870 2003 FEBS 2003 doi Site-directed mutagenesis of a loop at the active site of E1 a2p2 of the pyruvate dehydrogenase complex A possible common sequence motif Markus Fries Hitesh J. Chauhan Gonzalo J. Domingo Hyo-Il Jung and Richard N. Perham Cambridge Centre for Molecular Recognition Department of Biochemistry University of Cambridge UK Limited proteolysis of the pyruvate decarboxylase E1 a2b2 component of the pyruvate dehydrogenase PDH multienzyme complex of Bacillus stearothermophilus has indicated the importance for catalysis of a site Tyr281-Arg282 in the E1a subunit Chauhan . Domingo . Jung . Perham . 2000 Eur. J. Biochem. 267 7158-7169 . This site appears to be conserved in the a-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 E1a 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 . Keywords pyruvate dehydrogenase multienzyme complex thiamin diphosphate limited proteolysis enzyme mechanism. The family of 2-oxo acid dehydrogenase 2-OADH multienzyme complexes contains three members the pyruvate dehydrogenase PDH the 2-oxoglutarate dehydrogenase OGDH and the branched-chain 2-oxo acid dehydrogenase .