| Abstract
| - Bacterial luciferase is a heterodimeric (αβ) enzyme which catalyzes a light-producing reactionin Vibrio harveyi. In addition to the αβ enzyme, the β subunit can self-associate to form a stable butinactive homodimer [Sinclair, J. F., Ziegler, M. M., and Baldwin, T. O. (1994) Nat. Struct. Biol.1, 320−326]. The studies reported here were undertaken to explore the role of the subunit interface in theconformational stability of the enzyme. To this end, we constructed four mutant heterodimers in whichresidues at the subunit interface were changed in an effort to alter the volume of an apparent solventaccessible channel at the interface or to alter H-bonding groups. Equilibrium unfolding data for theheterodimer have been interpreted in terms of a three-state mechanism [Clark, C. A., Sinclair, J. F., andBaldwin, T. O. (1993) J. Biol. Chem. 268, 10773−10779]. However, we found that unfolding for thewild-type and mutant luciferases is better described by a four-state model. This change in the proposedmechanism of unfolding is based on observation of residual structure in the subunits following dissociationof the heterodimeric intermediate. All of the mutants display modest reductions in activity but, surprisingly,no change in the ΔG2H2O value for subunit dissociation and no measurable change in the equilibriumdissociation constant relative to that of the wild-type heterodimer. However, the ΔG1H2O value for theformation of the dimeric intermediate that precedes subunit dissociation is reduced for three of the mutants,indicating that mutations at the interface can alter the stability of a region of the α subunit that is distantfrom the interface. We conclude that the interface region communicates with the distal domains of thissubunit, probably through the active center region of the enzyme.
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