CATALYTICALLY ACTIVE FORMS OF THE INDIVIDUAL SUBUNITS OF VIBRIO-HARVEYI LUCIFERASE AND THEIR KINETIC AND BINDING-PROPERTIES

Contradictory findings have recently been reported regarding the (in)a bilities of individual subunits of the Vibrio harveyi alpha beta dimer ic luciferase to catalyze bioluminescence. We have produced individual alpha and beta subunits separately in Escherichia coli JM109 cells by recombinant DNA techniques. Both subunits were purified to more than 90% homogeneity and found to be catalytically active, with their gener al catalytic properties and the specific activities similar to those r eported earlier (Sinclair, J. F., Waddle, J. J., Waddill, E. F., and B aldwin, T. O. (1993) Biochemistry 32, 5038-5044). Individual subunits were significantly distinct from the native luciferase with respect to inactivations by trypsin and N-ethylmaleimide, and the stability of t he flavin 4a-hydroperoxide intermediate. The active species in isolate d alpha and beta samples were each the predominant protein species, co rresponding to a 42,000 M(r) alpha monomer and a 67,000 M(r) beta dime r, respectively. These findings clearly indicate that the activities o f the individual subunits are not due to trace contaminations of the r espective counter subunits. The much reduced specific activities of th e individual subunits are, in part, a consequence of diminished abilit ies to oxidize the aldehyde substrate. Kinetic and equilibrium measure ments indicate that alpha and beta(2) each contained a reduced flavin site, an aldehyde substrate site, and an aldehyde inhib itor site. The on and off rates of the decanal inhibitor binding were substantially slower than the bindings of decanal and reduced riboflavin 5'-phosphat e substrates. These findings are consistent with a scheme that the ald ehyde inhibitor blocks the binding of the reduced flavin substrate.