Mechanism of aldehyde inhibition of Vibrio harveyi luciferase. Identification of two aldehyde sites and relationship between aldehyde and flavin binding.
Vibrio harveyi luciferase is sensitive to aldehyde substrate inhibition, and two kinetic schemes have been previously postulated to account for such an inhibition. One scheme depicts a sequential binding of 2 aldehyde molecules, yielding an active enzyme-aldehyde binary complex and subsequently an inactive enzyme-(aldehyde)2 ternary complex (Holzman, T. F., and Baldwin, T. O. (1983) Biochemistry 22, 2838-2846). This two-aldehyde model was later withdrawn, and recently, a different scheme was proposed, following which the prior binding of one aldehyde to the native luciferase forms an inactive dead-end complex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 equilibrium studies were carried out to elucidate further the mechanism of aldehyde inhibition. Two, presumably independent, aldehyde-binding sites were detected, with a higher affinity site for the aldehyde substrate 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, whereas the binding of the decanal substrate enhances the affinity of the reduced riboflavin 5'-phosphate (FMNH2) site, the binding of decanal to the inhibitor site competes against FMNH2 binding, thus resulting in inhibition of luciferase activity. These findings are not compatible with either of the two earlier schemes mentioned above. A new kinetic model is formulated for the mechanism of aldehyde inhibition. Theoretical kinetic behaviors predicted on the basis of this model are in excellent agreement with experimental observations. A particularly reactive cysteine (residue 106) on the alpha subunit has been previously demonstrated 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 indicate that this residue is at or near the aldehyde inhibitor site. Relative locations of this residue and binding sites for FMNH2, the aldehyde substrate, and the aldehyde inhibitor are proposed.
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Department of Biochemical Chemistry, University of Houston, Texas 77204-5934.
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D000447:Aldehydes / Q000378:metabolism / Q000494:pharmacology*; D001667:Binding, Competitive; D003545:Cysteine / Q000378:metabolism; D005415:Flavins / Q000378:metabolism*; D007700:Kinetics; D008156:Luciferases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D014733:Vibrio / Q000201:enzymology*
D000447:Aldehydes; D005415:Flavins; D008156:Luciferases; D003545:Cysteine