Identification of the histidine residue in Escherichia coli isocitrate lyase that reacts with diethylpyrocarbonate.
Escherichia coli isocitrate lyase was inactivated by diethylpyrocarbonate in a pseudo-first-order process. The enzyme was completely inactivated by modification of a single histidine residue, but slower modification of further residues also occurred. The substrate, isocitrate, and products, glyoxylate and succinate, protected against inactivation by diethylpyrocarbonate but this was not simply due to binding at the active site. Treatment of the inactivated enzyme with hydroxylamine led to only partial recovery of activity. Diethylpyrocarbonate also reacted with sulphydryl groups in isocitrate lyase, as judged by titrations with Nbs2, but this reaction was not responsible for the failure of hydroxylamine to reactivate the enzyme fully. The reactivity of isocitrate lyase to diethylpyrocarbonate declined with pH, following a titration curve for a group of pKa 6.1. Isolation and sequencing of ethoxyformylated peptides showed that the major site of modification by diethylpyrocarbonate was histidine residue 306.
Rua
J
J
Department of Biochemistry, University of Glasgow, UK.
Robertson
A G
AG
Nimmo
H G
HG
D000595:Amino Acid Sequence; D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D004793:Enzyme Reactivators / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007522:Isocitrate Lyase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D007523:Isocitrates / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D013438:Sulfhydryl Compounds; D014357:Trypsin
D004793:Enzyme Reactivators; D006898:Hydroxylamines; D007523:Isocitrates; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D019811:Hydroxylamine; D006639:Histidine; C034219:isocitric acid; D014357:Trypsin; D007522:Isocitrate Lyase; D004047:Diethyl Pyrocarbonate