Site-directed mutagenesis of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Anacystis nidulans.
Using oligonucleotide-directed mutagenesis of the gene encoding the small subunit (rbcS) from Anacystis nidulans mutant enzymes have been generated with either Trp-54 of the small subunit replaced by a Phe residue, or with Trp-57 replaced by a Phe residue, whereas both Trp-54 and Trp-57 have been replaced by Phe residues in a double mutant. Trp-54 and Trp-57 are conserved in all amino acid sequences or the small subunit (S) that are known at present. The wild-type and mutant forms of Rubisco have all been purified to homogeneity. The wild-type enzyme, purified from Escherichia coli is indistinguishable from enzyme similarly purified from A. nidulans in subunit composition, subunit molecular mass and kinetic parameters (Vmax CO2 = 2.9 U/mg, Km CO2 = 155 microM). The single Trp mutants are indistinguishable from the wild-type enzyme by criteria (a) and (b). However, whereas, Km CO2 is also unchanged, Vmax CO2 is 2.5-fold smaller than the value for the wild-type enzyme for both mutants, demonstrating for the first time that single amino acid replacements in the non-catalytic small subunit influence the catalytic rate of the enzyme. The specificity factor tau, which measures the partitioning of the active site between the carboxylase and oxygenase reactions, was found to be invariant. Since tau is not affected by these mutations we conclude that S is an activating not a regulating subunit.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002245:Carbon Dioxide; D000458:Cyanobacteria / Q000201:enzymology*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008433:Mathematics; D009154:Mutation; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics*
D000596:Amino Acids; D004269:DNA, Bacterial; D002245:Carbon Dioxide; D004262:DNA Restriction Enzymes; D012273:Ribulose-Bisphosphate Carboxylase