KINETIC MECHANISM OF LUCIFERASE SUBUNIT FOLDING AND ASSEMBLY

The kinetic mechanism in vitro of the folding and assembly of the hete rodimeric flavin monooxygenase bacterial luciferase has been defined b y a unique set of rate constants which describe both the productive re folding pathway and competing off-pathway reactions in 50 mM phosphate , pH 7.0 at 18 degrees C. The individual alpha and beta subunits fold independently to form heterodimerization-competent species, alpha(i) a nd beta(i). The alpha(i) and beta(i) species can interact to form an i nactive heterodimeric intermediate, [alpha beta], which isomerizes to form the active alpha beta structure; the structure of the enzyme has been determined to 1.5 Angstrom resolution [Fisher, A. J., Thompson, T . B., Thoden, J. B., Baldwin, T. O., & Rayment, I. (1996) J. Biol. Che m. 271, 21956-21968]. In the absence of alpha(i), beta(i) can form a k inetically trapped homodimer, Pt, with a second-order rate constant of about 180 M(-1) s(-1) [Sinclair, J. F., Ziegler, M. M., & Baldwin, T. O. (1994) Not. Struct. Biol. 1, 320-326]; the structure of beta(2) ha s recently been reported [Thoden, J. B., Holden, H. M., Fisher, A. J., Sinclair, J. F., Wesenberg, G., Baldwin, T. O., & Rayment, I. (1997) Protein Sci. 6, 13-23]. The beta(i) species, or some other form that p recedes beta(i) on the refolding pathway, can also undergo a first-ord er conversion into a form (designated beta(x)) that cannot associate w ith alpha(i) to form the native enzyme. The rate constant for this pro cess, assigned here, accounts well for the previously observed depende nce of final yield on concentration of refolding species [Ziegler, M. M., Goldberg, M. E., Chaffotte, A. F., & Baldwin, T. O. (1993) J. Biol . Chem. 268, 10760-10765]. In simulations of the refolding reaction, a ll processes associated with the refolding of the individual subunits were combined into single first-order rate constants for each subunit which were consistent with the rate constants determined from stopped- flow circular dichroism studies. The first-order rate constant for the folding of the alpha subunit, estimated from the concentration-indepe ndent lag preceding the appearance of active enzyme, and the second-or der rate constant for assembly of alpha(i) and beta(i) into the hetero dimer, estimated from the concentration-dependent rate of appearance o f active enzyme, were consistent with the rates of first- and second-o rder processes monitored by changes in fluorescence of an extrinsic pr obe [the product of modification with N-(4-anilino-1-naphthyl)maleimid e] on the alpha subunit during refolding. The rate constant for the is omerization of [alpha beta](I) to form the active heterodimer was esti mated from the kinetic data of a secondary dilution experiment and fro m fluorescence measurements of protein diluted 20-fold from 2.1 M urea -containing buffer. The rate constants reported here for the kinetic m echanism of refolding permitted simulation of the time courses and yie lds for activity recovery during the refolding of luciferase from abou t 1 to 25 mu g/mL which are in excellent agreement with our previously reported data.