title: The catalytic domain of dihydrolipoyl acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Expression, purification and reversible denaturation.
abstract: A sub-gene encoding the catalytic (acetyltransferase) domain (E2pCD) comprising residues 173-427 of the dihydrolipoyl acetyltransferase (E2p) chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was expressed in Escherichia coli. The product assembled to form the characteristic icosahedral (60-mer) core structure with full catalytic activity. The Km values for dihydrolipoamide and acetyl-CoA were 1.2 mM and 13 microM, respectively. Dissociation of the icosahedral E2pCD into monomers by exposure to guanidine hydrochloride and the subsequent reassociation by gradual removal of the denaturing agent demonstrated the ability of the polypeptide chain to fold and reassemble in the absence of chaperonins.
authors.affiliation: Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, UK.
authors.forename: M D
authors.initials: MD
authors.lastname: Allen
authors.forename: R N
authors.initials: RN
authors.lastname: Perham
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000123:Acetyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D002384:Catalysis; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D004926:Escherichia coli / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry*; D011993:Recombinant Fusion Proteins; D008063:Thioctic Acid / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D006146:Guanidines; D011768:Pyruvate Dehydrogenase Complex; D011993:Recombinant Fusion Proteins; C007409:dihydrolipoamide; D000105:Acetyl Coenzyme A; D008063:Thioctic Acid; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D019791:Guanidine