CONTRIBUTION OF FOLDING STEPS INVOLVING THE INDIVIDUAL SUBUNITS OF BACTERIAL LUCIFERASE TO THE ASSEMBLY OF THE ACTIVE HETERODIMERIC ENZYME

Bacterial luciferase is an alphabeta heterodimer with a single active center in which the reaction of reduced FMN, O2, and an aliphatic alde hyde yields a photon of blue-green light. We have shown that refolding of the luciferase subunits from 5 M urea occurs via the intermediacy of several species, one of which is an inactive heterodimeric structur e, resulting from the dimerization of alpha and beta, which isomerizes to the active alphabeta structure in a first-order reaction (Ziegler, M. M., Goldberg, M. E., Chaffotte, A. F., and Baldwin, T. O. (1993) J . Biol. Chem. 268, 10760-10765). We have also demonstrated the existen ce of an inactive heterodimeric species that is well populated at equi librium in the presence of 1.6-2.8 M urea (Clark, A. C., Sinclair, J. F., and Baldwin, T. O. (1993) J. Biol. Chem. 268, 10773-10779). We hav e separated the alpha and beta subunits by ion exchange chromatography and investigated the effects on reformation of active luciferase of a llowing the individual subunits to refold separately prior to mixing. These investigations show that the lag in formation of active lucifera se is due to slow steps in folding of the individual subunits. The bet a subunit appears to fold faster than the alpha subunit, but folding o f the beta subunit also shows a distinct lag. When the alpha and beta subunits were allowed to refold from urea for periods of several hours or more prior to mixing, the yield of active heterodimeric luciferase was compromised, which is consistent with the finding that individual subunits produced in vivo fold into structures incompetent to interac t with each other to form the active heterodimer (Waddle, J. J., Johns ton, T. C., and Baldwin, T. O. (1987) Biochemistry 26, 4917-4921). It appeared that the rate with which the beta subunit assumed the heterod imerization-incompetent structure was faster than the rate with which the alpha subunit became heterodimerization-incompetent. These observa tions support a model for folding and assembly of the subunits of luci ferase in which the two subunits fold into assembly-competent structur es that associate to form the heterodimer. In a slow competing process , the subunits undergo a conformational rearrangement to form stable s tructures incompetent to form heterodimers. It appears that the associ ation of the luciferase subunits might constitute an example of one po lypeptide modifying the folding pathway of another, a model that is co nsistent with the suggestion that the formation of the heterodimeric s tructure of luciferase is a kinetic trap on the folding pathway of the individual subunits (Sugihara, J., and Baldwin, T. O. (1988) Biochemi stry 27, 2872-2880).