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J. Biol. Chem., Vol. 281, Issue 35, 25241-25249, September 1, 2006

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Increased Sensitivity of Oxidized Large Isoform of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase to ADP Inhibition Is Due to an Interaction between Its Carboxyl Extension and Nucleotide-binding Pocket^*

Dafu Wang and Archie R. Portis, Jr.¹

From the Department of Plant Biology, University of Illinois, Urbana, Illinois 61801 and the Photosynthesis Research Unit, Agricultural Research Service, United States Department of Agriculture, Urbana, Illinois 61801

In Arabidopsis, oxidation of the large (46-kDa) isoform activase to form a disulfide bond in the C-terminal extension (C-extension) significantly increases its ADP sensitivity for both ATP hydrolysis and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation, thereby decreasing both activities at physiological ratios of ADP/ATP. In this study, we demonstrate that the C-extension of the oxidized large activase isoform can be cross-linked with regions containing residues that contribute to the nucleotide-binding pocket, with a higher efficiency in the presence of ADP or the absence of nucleotides than with ATP. Coupled with measurements demonstrating a redox-dependent protease sensitivity of the C-extension and a lower ATP or adenosine 5'-O-(thiotriphosphate) (ATPS) affinity of the oxidized large isoform than either the reduced form or the smaller isoform, the results suggest that the C-extension plays an inhibitory role in ATP hydrolysis, regulated by redox changes. In contrast, the ADP affinities of the small isoform and the reduced or oxidized large isoform were similar, which indicates that the C-extension selectively interferes with the proper binding of ATP, possibly by interfering with the coordination of the -phosphate. Furthermore, replacement of conserved, negatively charged residues (Asp³⁹⁰, Glu³⁹⁴, and Asp⁴⁰¹) in the C-extension with alanine significantly reduced the sensitivities of the mutants to ADP inhibition, which suggests the involvement of electrostatic interactions between them and positively charged residues in or near the nucleotide-binding pocket. These studies provide new insights into the mechanism of redox regulation of activase by the C-extension in the large isoform.

Received for publication, May 17, 2006 , and in revised form, June 22, 2006.

^* This work was supported in part by a grant from the United States Department of Energy (DE-AI02-97ER20268). 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.

¹ To whom correspondence should be addressed. Tel.: 217-244-3083; Fax: 217-244-4419; E-mail: arportis{at}uiuc.edu.

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