Examination of the intersubunit interaction between glutamate-48 and lysine-168 of ribulose-bisphosphate carboxylase/oxygenase by site-directed mutagenesis.
The active site of ribulose-bisphosphate carboxylase/oxygenase is constituted from domains of adjacent subunits and includes an intersubunit electrostatic interaction between Lys 168 and Glu48, which has been recently identified by x-ray crystallography (Andersson, I., Knight, S., Schneider, G., Lindqvist, Y., Lundqvist, T., Brändén, C.-I., and Lorimer, G.H. (1989) Nature 337, 229-234; Lundqvist, T., and Schneider, G. (1989) J. Biol. Chem. 264, 7078-7083). To examine the structural and functional requirements for this interaction, we have used site-directed mutagenesis to replace Lys168 of the homodimeric enzyme from Rhodospirillum rubrum with arginine, glutamine, or glutamic acid. All three substitutions result in mutant enzymes with less than or equal to 0.1% of wild-type activity. The nonconservative substitution of Lys168 with a glutamyl residue precludes the formation of a stable dimer, explaining the consequential abolition of enzymic activity. Both the Arg168 and Gln168 mutant proteins are isolated as stable dimers, even though the latter obviously lacks an electrostatic interaction present in the wild-type enzyme. Despite the absence of overall carboxylase activity, these two mutant proteins serve as catalysts for the enolization of ribulose bisphosphate, as measured by exchange of the C3 proton with solvent. These observations, as well as ligand-binding properties of the mutant proteins, are consistent with Lys168 facilitating a catalytic step subsequent to enolization.
Mural
R J
RJ
Protein Engineering and Molecular Mutagenesis Program, Oak Ridge National Laboratory, Tennessee 37831-8077.
Soper
T S
TS
Larimer
F W
FW
Hartman
F C
FC
D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D012247:Rhodospirillum rubrum / Q000201:enzymology* / Q000235:genetics; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism*
D004269:DNA, Bacterial; D005971:Glutamates; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D018698:Glutamic Acid; D012273:Ribulose-Bisphosphate Carboxylase; D008239:Lysine