title: Catalytically active forms of the individual subunits of Vibrio harveyi luciferase and their kinetic and binding properties.
abstract: Contradictory findings have recently been reported regarding the (in)abilities of individual subunits of the Vibrio harveyi alpha beta dimeric 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 general catalytic properties and the specific activities similar to those reported earlier (Sinclair, J. F., Waddle, J. J., Waddill, E. F., and Baldwin, T. O. (1993) Biochemistry 32, 5036-5044). Individual subunits were significantly distinct from the native luciferase with respect to inactivations by trypsin and N-ethylmaleimide, and the stability of the flavin 4a-hydroperoxide intermediate. The active species in isolated alpha and beta samples were each the predominant protein species, corresponding to a 42,000 M(r) alpha monomer and a 67,000 M(r) beta dimer, respectively. These findings clearly indicate that the activities of the individual subunits are not due to trace contaminations of the respective counter subunits. The much reduced specific activities of the individual subunits are, in part, a consequence of diminished abilities to oxidize the aldehyde substrate. Kinetic and equilibrium measurements indicate that alpha and beta 2 each contained a reduced flavin site, an aldehyde substrate site, and an aldehyde inhibitor site. The on and off rates of the decanal inhibitor binding were substantially slower than the bindings of decanal and reduced riboflavin 5'-phosphate substrates. These findings are consistent with a scheme that the aldehyde inhibitor blocks the binding of the reduced flavin substrate.
authors.affiliation: Department of Biochemical Sciences, University of Houston, Texas 77204-5934, USA.
authors.forename: H
authors.initials: H
authors.lastname: Choi
authors.forename: C K
authors.initials: CK
authors.lastname: Tang
authors.forename: S C
authors.initials: SC
authors.lastname: Tu
mesh_terms: D001665:Binding Sites; D004795:Enzyme Stability; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry / Q000378:metabolism*; D014733:Vibrio / Q000201:enzymology*
chemical_list: D008156:Luciferases