The amino acid sequence encompassing the active-site histidine residue of lipoamide dehydrogenase from Escherichia coli labelled with a bifunctional arsenoxide.
Pyruvate dehydrogenase multienzyme complex (PD complex) in the presence of pyruvate, thiamine pyrophosphate, coenzyme A, and Mg2+ (or NADH) was irreversibly inhibited with the radiolabelled bifunctional aresenoxide p-[(bromoacetyl)amino]phenyl arsenoxide (BrCH2 14CONHPhAsO). The initial reaction of the reagent was with a reduced lipoyl group of the lipoamide acetyltransferase component to form a dithioarsinite complex. Following the normal catalytic reactions, the anchored reagent was delivered into the active site of the lipoamide dehydrogenase (E3) component where an irreversible alkylation ensued via the bromoacetamidyl moiety. Treatment with 2,3-dithiopropanol (to break dithioarsinite bonds) caused the radiolabelled reagent to reside with E3. E3 was isolated from the inhibited PD complex and CNBr cleavage of the inhibited enzyme yielded a single radiolabelled peptide that was purified on a cyanopropyl silica column using high performance liquid chromatography. The radiolabelled amino acid was identified (after acid hydrolysis) as N3-[14C]carboxymethyl histidine in agreement with earlier studies. The radiolabel was located in residue 14 of the peptide for which the sequence was determined as GCDAEDIALTIHAHPTL-EIVGLAAEVFEG. This sequence agrees with the amino acid sequence determined from the gene sequence of E3. The histidine alkylated in the E3 component of the PD complex by BrCH2 14CONHPhAsO is residue-444 and further establishes its active site role.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001152:Arsenicals / Q000494:pharmacology*; D001665:Binding Sites; D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D008058:Dihydrolipoamide Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004912:Erythrocytes / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine; D006801:Humans; D009206:Myocardium / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D013045:Species Specificity; D013552:Swine
D000596:Amino Acids; D001152:Arsenicals; D002250:Carbon Radioisotopes; D010446:Peptide Fragments; D006639:Histidine; C031672:4-((bromoacetyl)amino)phenyl arsenoxide; D008058:Dihydrolipoamide Dehydrogenase; D003488:Cyanogen Bromide