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J. Biol. Chem., Vol. 282, Issue 49, 36068-36076, December 7, 2007

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Differences in Carbon Isotope Discrimination of Three Variants of D-Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Reflect Differences in Their Catalytic Mechanisms^*

Dennis B. McNevin, Murray R. Badger, Spencer M. Whitney, Susanne von Caemmerer, Guillaume G. B. Tcherkez^¶, and Graham D. Farquhar^||1

From the Molecular Plant Physiology and ^||Environmental Biology, Research School of Biological Sciences, The Australian National University, Canberra, and the School of Health Sciences, University of Canberra, Bruce, Australian Capital Territory 2601, Australia and the ^¶Laboratoire d'Ecophysiologie Végétale, Université Paris Sud XI, 91405 Orsay Cedex, France

The carboxylation kinetic (stable carbon) isotope effect was measured for purified D-ribulose-1,5-bisphosphate carboxylases/oxygenases (Rubiscos) with aqueous CO₂ as substrate by monitoring Rayleigh fractionation using membrane inlet mass spectrometry. This resulted in discriminations () of 27.4 ± 0.9 for wild-type tobacco Rubisco, 22.2 ± 2.1 for Rhodospirillum rubrum Rubisco, and 11.2 ± 1.6 for a large subunit mutant of tobacco Rubisco in which Leu³³⁵ is mutated to valine (L335V). These 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 values are indicative of the potential evolutionary variability of 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 values for the Rubisco catalytic mechanism and suggest that Rubiscos that are associated with a lower value have a less product-like carboxylation transition state and/or allow a decarboxylation step that evolution has excluded in higher plants.

Received for publication, July 30, 2007 , and in revised form, October 3, 2007.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental "Appendix."

This article was selected as a Paper of the Week.

¹ To whom correspondence should be addressed: Environmental Biology, Research School of Biological Sciences, The Australian National University, G.P.O. Box 475, Canberra, ACT 2601, Australia. Tel.: 61-2-6125-3743; Fax: 61-2-6125-4919; E-mail: graham.farquhar{at}anu.edu.au.

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