Amino acid substitutions in the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase that influence catalytic activity of the holoenzyme.
Four unique amino acid substitutions were introduced by site-directed mutagenesis into the third conserved region of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) from Anacystis nidulans (Synechococcus sp., PCC6301), resulting in the formation of four mutant enzymes, I87V, R88K, G91V, and F92L. Wild-type and mutant proteins were purified after synthesis in Escherichia coli. These single amino acid substitutions do not appear to perturb intersubunit interactions or induce any gross conformational changes; purified mutant proteins are stable, for the most part like the wild-type holoenzyme, and exhibit nearly identical CD spectra. Three of the four mutants, however, are severely deficient in carboxylase activity, with kcat less than or equal to 35% of the wild-type enzyme. While the substrate specificity factors were the same for the mutant and wild-type enzymes, significant alterations in some kinetic parameters were observed, particularly in the Michaelis constants for CO2, O2, and RuBP. All four mutant proteins exhibited lower KCO2 values, ranging from 37 to 88% of the wild-type enzyme. Two of the mutants, in addition, exhibited significantly lower KRuBP values, and one mutant showed a substantial decrease in KO2. The effects of the single-site mutations in rbcS of this study strengthen the hypothesis that small subunits may not contribute directly to substrate specificity; however, individual residues of the small subunit substantially influence catalysis by large subunits.
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Department of Microbiology, Ohio State University, Columbus 43210.
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002384:Catalysis; D000458:Cyanobacteria / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics* / Q000302:isolation & purification
D000596:Amino Acids; D001426:Bacterial Proteins; D012273:Ribulose-Bisphosphate Carboxylase