title: Essentiality of Lys-329 of ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum as demonstrated by site-directed mutagenesis.
abstract: The unusual chemical properties of active-site Lys-329 of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have suggested that this residue is required for catalysis. To test this postulate Lys-329 was replaced with glycine, serine, alanine, cysteine, arginine, glutamic acid or glutamine by site-directed mutagenesis. These single amino acid substitutions do not appear to induce major conformational changes because (i) intersubunit interactions are unperturbed in that the purified mutant proteins are stable dimers like the wild-type enzyme and (ii) intrasubunit folding is normal in that the mutant proteins bind the competitive inhibitor 6-phosphogluconate with an affinity similar to that of wild-type enzyme. In contrast, all of the mutant proteins are severely deficient in carboxylase activity (less than 0.01% of wild-type) and are unable to form the exchange-inert complex, characteristic of the wild-type enzyme, with the transition-state analogue carboxyarabinitol bisphosphate. These results underscore the stringency of the requirement for a lysyl side-chain at position 329 and imply that Lys-329 is involved in catalysis, perhaps stabilizing a transition state in the overall reaction pathway.
authors.affiliation: Biology Division, Oak Ridge National Laboratory, TN 37831.
authors.forename: T S
authors.initials: TS
authors.lastname: Soper
authors.forename: R J
authors.initials: RJ
authors.lastname: Mural
authors.forename: F W
authors.initials: FW
authors.lastname: Larimer
authors.forename: E H
authors.initials: EH
authors.lastname: Lee
authors.forename: R
authors.initials: R
authors.lastname: Machanoff
authors.forename: F C
authors.initials: FC
authors.lastname: Hartman
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D008239:Lysine; D008969:Molecular Sequence Data; D009154:Mutation; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics* / Q000378:metabolism
chemical_list: D012273:Ribulose-Bisphosphate Carboxylase; D008239:Lysine