Open AccessThe Reactivity of One Essential Cysteine as a Conformational Probe in Escherichia coli Tryptophanase
Author(s) -
RAIBAUD Olivier,
GOLDBERG Michel E.
Publication year - 1977
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1977.tb11354.x
Subject(s) - tryptophanase , escherichia coli , cysteine , chemistry , reactivity (psychology) , biochemistry , stereochemistry , medicine , enzyme , alternative medicine , pathology , gene
It is first shown that the inactivation of Escherichia coli apotryptophanase by N ‐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 a second, corresponding to active enzyme, where the cysteine is masked. The rate of the isomerization step involved in this change in conformation is measured by the stopped‐flow technique (τ= 0.4 s). 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.