Kinetic Mechanism of Luciferase Subunit Folding and Assembly

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Abstract
The kinetic mechanism in vitro of the folding and assembly of the heterodimeric flavin monooxygenase bacterial luciferase has been defined by a unique set of rate constants which describe both the productive refolding pathway and competing off-pathway reactions in 50 mM phosphate, pH 7.0 at 18 °C. The individual α and β subunits fold independently to form heterodimerization-competent species, αi and βi. The αi and βi species can interact to form an inactive heterodimeric intermediate, [αβ]I, which isomerizes to form the active αβ structure; the structure of the enzyme has been determined to 1.5 Å resolution [Fisher, A. J., Thompson, T. B., Thoden, J. B., Baldwin, T. O., & Rayment, I. (1996) J. Biol. Chem. 271, 21956−21968]. In the absence of αi, βi can form a kinetically trapped homodimer, β2, with a second-order rate constant of about 180 M-1 s-1 [Sinclair, J. F., Ziegler, M. M., & Baldwin, T. O. (1994) Nat. Struct. Biol. 1, 320−326]; the structure of β2 has 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 βi species, or some other form that precedes βi on the refolding pathway, can also undergo a first-order conversion into a form (designated βx) that cannot associate with αi to form the native enzyme. The rate constant for this process, assigned here, accounts well for the previously observed dependence 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, all 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 α subunit, estimated from the concentration-independent lag preceding the appearance of active enzyme, and the second-order rate constant for assembly of αi and βi into the heterodimer, estimated from the concentration-dependent rate of appearance of active enzyme, were consistent with the rates of first- and second-order processes monitored by changes in fluorescence of an extrinsic probe [the product of modification with N-(4-anilino-1-naphthyl)maleimide] on the α subunit during refolding. The rate constant for the isomerization of [αβ]I to form the active heterodimer was estimated from the kinetic data of a secondary dilution experiment and from fluorescence measurements of protein diluted 20-fold from 2.1 M urea-containing buffer. The rate constants reported here for the kinetic mechanism of refolding permitted simulation of the time courses and yields for activity recovery during the refolding of luciferase from about 1 to 25 μg/mL which are in excellent agreement with our previously reported data.

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