The Reactivity of One Essential Cysteine as a Conformational Probe in Escherichia coli Tryptophanase

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Abstract
The inactivation of E. coli apotryptophanase [EC 4.1.99.1] 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. The protein can exist under 2 classes of conformation: one, corresponding to inactive protein, in which the cysteine is reactive, and a 2nd, 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 (.tau. = 0.4 s). 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). This criterion shows that dimeric holotryptophanase falls in the class of inactive conformations. 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 are discussed.