title: Differences in carbon isotope discrimination of three variants of D-ribulose-1,5-bisphosphate carboxylase/oxygenase reflect differences in their catalytic mechanisms.
abstract: The carboxylation kinetic (stable carbon) isotope effect was measured for purified d-ribulose-1,5-bisphosphate carboxylases/oxygenases (Rubiscos) with aqueous CO(2) as substrate by monitoring Rayleigh fractionation using membrane inlet mass spectrometry. This resulted in discriminations (Delta) of 27.4 +/- 0.9 per thousand for wild-type tobacco Rubisco, 22.2 +/- 2.1 per thousand for Rhodospirillum rubrum Rubisco, and 11.2 +/- 1.6 per thousand for a large subunit mutant of tobacco Rubisco in which Leu(335) is mutated to valine (L335V). These Delta values are consistent with the photosynthetic discrimination determined for wild-type tobacco and transplastomic tobacco lines that exclusively produce R. rubrum or L335V Rubisco. The Delta values are indicative of the potential evolutionary variability of Delta values for a range of Rubiscos from different species: Form I Rubisco from higher plants; prokaryotic Rubiscos, including Form II; and the L335V mutant. We explore the implications of these Delta values for the Rubisco catalytic mechanism and suggest that Rubiscos that are associated with a lower Delta value have a less product-like carboxylation transition state and/or allow a decarboxylation step that evolution has excluded in higher plants.
authors.affiliation: Molecular Plant Physiology and Environmental Biology, Research School of Biological Sciences, The Australian National University, Canberra, Australian Capital Territory, Australia.
authors.forename: Dennis B
authors.initials: DB
authors.lastname: McNevin
authors.forename: Murray R
authors.initials: MR
authors.lastname: Badger
authors.forename: Spencer M
authors.initials: SM
authors.lastname: Whitney
authors.forename: Susanne
authors.initials: S
authors.lastname: von Caemmerer
authors.forename: Guillaume G B
authors.initials: GG
authors.lastname: Tcherkez
authors.forename: Graham D
authors.initials: GD
authors.lastname: Farquhar
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002245:Carbon Dioxide / Q000737:chemistry / Q000378:metabolism; D002247:Carbon Isotopes / Q000737:chemistry; D002384:Catalysis; D020125:Mutation, Missense; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D012247:Rhodospirillum rubrum / Q000201:enzymology* / Q000235:genetics; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D002247:Carbon Isotopes; D010940:Plant Proteins; D002245:Carbon Dioxide; D012273:Ribulose-Bisphosphate Carboxylase