REVERSIBLE DISSOCIATION AND CONFORMATIONAL STABILITY OF DIMERIC RIBULOSE BISPHOSPHATE CARBOXYLASE

Dimer-monomer dissociation of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum was investigated using hydrostatic pressure in the range 1-2 kbar to promote dissociation. Intrinsic fluorescence emission and polarization, along with the polarization of the fluorescence of single-labeled AEDANS conjugates, were used to follow the dissociation. Full reversibility after dissociation was observed to depend on the presence of small ligands: glycerol, Mg2+, and NaHCO3, the last two being required to activate the enzyme. The free energy of association at 15-degrees-C, -12.9 kcal mol-1, was made up of a positive change in enthalpy on association of 6.0 kcal mol-1 and an entropic contribution (TDELTAS) of 18.9 kcal mol-1; thus the monomer association is entropy driven. No dissociation of the quaternary complex formed by the dimer, 2-carboxy-D-arabinitol 1,5-diphosphate (CADP), Mg2+, and NaHCO3 was observed at pressures up to 2.0 kbar; the magnitude of stabilization by the inhibitor binding was estimated as 2.3 kcal mol-1. Pressurization in the presence of bis-ANS results in a time-dependent increase in fluorophore emission, indicating changes in monomer conformation with exposure of hydrophobic surfaces upon dissociation. Reactivity against the fluorescent probe 1,5-I-AEDANS was also used as a conformational probe: HPLC of a trypsin digest of rubisco labeled at atmospheric pressure revealed a single fluorescent peptide, whereas more extensive labeling was observed when the reaction was carried out at 2.0 kbar, indicative of exposure of internal cysteines. Most of the properties of the monomeric species detected at high hydrostatic pressure, namely, the red-shifted tryptophan emission relative to the native dimer, binding of hydrophobic probes, and instability at high concentrations and at temperatures above 15-degrees-C, suggest those of the previously described folding intermediate (van der Vies, S. M., Viitanen, P. V., Gatenby, A. A., Lorimer, G. H., & Jaenicke, R. (1992) Biochemistry 31, 3635-3644). Further information about intersubunit affinity was obtained by reexamination of the enzyme carrying a single mutation, K168E (Mural, R. J., Soper, T. S., Larimer, F. W., & Hartman, F. (1990) J. Biol. Chem. 265, 6501-6505), previously described as a ''folded monomer''. By using size-exclusion HPLC, dilution, and fluorescence polarization at high pressure, we demonstrate that although the mutant shows a decrease in dissociation constant of 2 orders of magnitude compared with the wild-type enzyme, it is still able to form stable dimers.