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ARS Home » Midwest Area » Urbana, Illinois » Global Change and Photosynthesis Research » Research » Publications at this Location » Publication #197250
Title: INCREASED SENSITIVITY OF OXIDIZED LARGE ISOFORM OF RUBISCO ACTIVASE TO ADP INHIBITION IS DUE TO AN INTERACTION BETWEEN ITS CARBOXYL-EXTENSION AND NUCLEOTIDE-BINDING POCKET

Author
item WANG, DAFU - UNIVERSITY OF ILLINOIS
item Portis Jr, Archie

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/22/2006
Publication Date: 9/1/2006
Citation: Wang, D., Portis Jr, A.R. 2006. Increased sensitivity of oxidized large isoform of rubisco activase to adp inhibition is due to an interaction between its carboxyl-extension and nucleotide-binding pocket. Journal of Biological Chemistry. 281:25241-25249.

Interpretive Summary: The activity of Rubisco, the enzyme that captures carbon dioxide, often limits photosynthesis, the process by which plants use light energy from the sun to make carbohydrates for growth from carbon dioxide and water. Rubisco activity is maintained and regulated by another chloroplast protein, Rubisco activase. Rubisco activity might be increased to improve plant growth by altering the regulation of the activase. Previous work has shown that activase is regulated via an extended amino acid sequence present in one of two forms of the protein. In this work, we used site-directed mutagenesis of residues in this region and cross-linking with mass spectroscopy analysis to provide support for the hypothesis that this region interacts with the active site to modify its properties. This information will benefit scientists attempting to modify the properties of the activase in ways beneficial for increased photosynthesis by crop plants.

Technical Abstract: In Arabidopsis, oxidation of the large (46-kDa) isoform activase to form a disulfide bond in the carboxyl-terminal extension (C-extension) significantly increases its ADP sensitivity for both ATP hydrolysis and Rubisco activation, thereby decreasing both activities at physiological ratios of ADP/ATP. In this communication, 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 no nucleotides or ADP than with ATP. Coupled with measurements demonstrating a redox-dependent protease sensitivity of the C-extension and a lower ATP (or ATP-'-S) 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 catalysis, 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 (D390, E394 and D401) 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.