Bacterial luciferase: demonstration of a catalytically competent altered conformational state following a single turnover

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Abstract
Ziegler-Nicoli et al. reported that a highly reactive cysteinyl residue on the .alpha. subunit of bacterial luciferase resides in or near the flavin binding site such that the enzyme-flavin complex is protected from inactivation by alkylating reagents. These authors also observed that injection of FMNH2 into an air-equilibrated solution of enzyme protected the enzyme from alkylation for much longer than the lifetime of the 4a-peroxydihydroflavin intermediate resulting from reaction of enzyme-bound FMNH2 with O2. Two related explanations were offered: either the product FMN dissociated from the enzyme much more slowly following a catalytic cycle than would be predicted from the Kd measured by equilibrium binding or the enzyme itself, without bound flavin, was in an altered conformational state in which the thiol was less reactive following a catalytic cycle. Either explanation involves a slow return of the enzyme to its initial state following a catalytic cycle. This phenomenon was investigated in more detail and it was found that rapid removal of the flavin from the enzyme by chromatography following catalytic turnover did not return the enzyme to its original state of susceptibility to either alkylating reagents or proteolytic enzymes. The flavin-free enzyme returned to the susceptible conformation with a half-time of .apprx. 25 min at 0.degree. C. Inactivation of the enzyme at intermediate times of relaxation by either a proteolytic enzyme or an alkylating reagent showed biphasic kinetics, indicative of a mixture of the protected and susceptible forms. These results demonstrate that the enzyme is in an altered conformational state following a single catalytic cycle and that this altered conformational state slowly relaxes to a conformer resembling the original in susceptibility to alkylating reagents and proteolytic enzymes.