The reactivity of one essential cysteine as a conformational probe in Escherichia coli tryptophanase. Application to the study of the structural influence of subunit interactions.
It is first shown that the inactivation of Escherichia coli apotryptophanase byN-ethylmaleimide results from the labeling of a single particularly reactive cysteine per protomer. The reactivity of this cysteine under various conditions is investigated and the results indicate that the protein can exist under two classes of conformation: one, corresponding to inactive protein,in which the cysteine is reactive, and second, corresponding to active enzyme, where the cysteine is masked. The rate of the isomeriation step involved in this change in conformation is measured by the stopped-flow technique(tou = 0.4s). Finally, the reactivity of the cysteine is used to characterize the conformation of dimeric holotryptophanase (i.e. a dissociated form of the enzyme obtained as a transient species between dimeric apoenzyme and the natural tetrameric holoenzyme). By this criterion, it is shown that dimeric holotryptophanase falls in the class of 'inactive' conformations. These results are used to discuss the influence of the quaternary structure on the functional and conformational properties of tryptophanase and the nature of the conformational change involved in the activation of the enzyme by its cofactor and specific cations.
D001059:Apoproteins / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008190:Lyases / Q000378:metabolism; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000032:analysis; D011188:Potassium / Q000494:pharmacology; D011485:Protein Binding; D012964:Sodium / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014357:Trypsin; D014368:Tryptophanase / Q000378:metabolism
D001059:Apoproteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D012964:Sodium; D014357:Trypsin; D008190:Lyases; D014368:Tryptophanase; D003545:Cysteine; D005033:Ethylmaleimide; D011188:Potassium