Examination of subunit interactions at the active site of ribulose 1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum by hybridization of site-directed mutants.

The two active sites of homodimeric ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum are constituted by interacting domains of adjacent subunits, in which residues from each are required for catalytic activity. Active-site residues include Lys-166 of one domain and Glu-48 of the interacting domain from the adjacent subunit. Whereas all substitutions for Lys-166, introduced by site-directed mutagenesis, abolished catalytic activity, only a negatively charged residue (e.g., aspartic acid) resulted in the disruption of the subunit interactions (Lee et al., 1987). This disruption could result from improper folding of the individual polypeptide chains or to more localized effects (e.g., charge-charge repulsion due to proximal negative charges of Asp-166 and Glu-48 of adjacent domains or conformational changes restricted to a single domain). To address these questions, we have examined the ability of the Asp-166 mutant subunit to associate with a mutant subunit in which the negatively charged Glu-48 has been replaced by the neutral glutaminyl residue. Coexpression in Escherichia coli of the genes for both mutant subunits results in formation of a catalytically active hybrid, despite the absence of activity when either gene is expressed individually. Isolation and characterization of the hybrid show that it is composed of one Asp-166 subunit and one Gln-48 subunit, presumably with only one functional active site per dimeric molecule. This association of dissimilar subunits shows that introduction of a negative charge at position 166 does not lead to overall distortion of subunit conformation. In contrast to the wild-type enzyme, the hybrid dissociates spontaneously at low protein concentration but is stabilized by elevated ionic strengths or by glycerol.