MECHANISM OF ALDEHYDE INHIBITION OF VIBRIO-HARVEYI LUCIFERASE - IDENTIFICATION OF 2 ALDEHYDE SITES AND RELATIONSHIP BETWEEN ALDEHYDE AND FLAVIN BINDING

Vibrio harveyi luciferase is sensitive to aldehyde substrate inhibitio n, and two kinetic schemes have been previously postulated to account for such an inhibition, One scheme depicts a sequential binding of 2 a ldehyde molecules, yielding an active enzyme-aldehyde binary complex a nd subsequently an inactive enzyme-(aldehyde)(2) ternary complex (Holz man, T. F., and Baldwin, T. O. (1983) Biochemistry 22, 2838-2846). Thi s two-aldehyde model was later withdrawn, and recently, a different sc heme was proposed, following which the prior binding of one aldehyde t o the native luciferase farms an inactive dead-end complex (Abu-Soud, H. M., Clark, A. C., Francisco, W. A, Baldwin, T. O., and Raushel, F.M . (1993) J. Biol. Chem. 268, 7699-7706). In this work, kinetic and equ ilibrium studies were carried out to elucidate further the mechanism o f aldehyde inhibition. Two, presumably independent, aldehyde-binding s ites were detected, with a higher affinity site for the aldehyde subst rate and a weaker affinity site for the aldehyde inhibitor. Binding to and dissociation from the inhibitor site by decanal were revealed by chemical relaxation analysis to be slow processes. Furthermore, wherea s the binding of the decanal substrate enhances the affinity of the re duced riboflavin 5'-phosphate (FMNH(2)) site, the binding of decanal t o the inhibitor site competes against FMNH(2) binding, thus resulting in inhibition of luciferase activity. These findings are not compatibl e with either of the two earlier schemes mentioned above. A new kineti c model is formulated for the mechanism of aldehyde inhibition. Theore tical kinetic behaviors predicted on the basis of this model are in ex cellent agreement with experimental observations. A particularly react ive cysteine (residue 106) on the alpha subunit has been previously de monstrated to be at or near an aldehyde site (Fried, A., and Tu, S.-C. (1984) J. Biol. Chem. 259, 10754-10759). Evidence is presented to ind icate that this residue is at or near the aldehyde inhibitor site. Rel ative locations of this residue and binding sites for FMNH(2), the ald ehyde substrate, and the aldehyde inhibitor are proposed.