Site-specific mutations in a loop region of the C-terminal domain of the large subunit of ribulose bisphosphate carboxylase/oxygenase that influence substrate partitioning.
Amino acids composing a flexible loop (loop 6) of the eight-stranded barrel domain of the L-subunit of Synechococcus ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) involved in reaction intermediate stabilization have been modified by site-specific mutagenesis. Changes at positions both distant and within the active site affect overall catalysis and substrate partitioning. Most significantly, replacement of the active site Lys (Lys-334) with Arg at the apex of the loop almost completely suppressed the carboxylase activity of the enzyme relative to oxygenation, with only a modest reduction in overall catalysis. Val-331 and Thr-342, more distant from the active site but with interacting side chains, were changed to larger and smaller residues with differential effects on both turnover and substrate partitioning. Substitution of the loop with the sequence found in more efficient carboxylases only increased partitioning marginally when accompanied by alterations in the C-terminal tail of the L-subunit that interacts with the loop. Generally, modifications to the loop composition also affected enediol formation, the first step of catalysis, suggesting that the geometry and hence flexibility of this segment affect more than just stabilization of the intermediates immediately following reaction with CO2 or O2.
Gutteridge
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Central Research and Development Department, Du Pont Company, Wilmington, Delaware 19880-0402.
Rhoades
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Herrmann
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D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010944:Plants / Q000201:enzymology; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000201:enzymology; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012273:Ribulose-Bisphosphate Carboxylase