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Title: ALTERED ATP-BINDING AND ATPASE ACTIVITY IN RUBISCO ACTIVASE MUTANTS

Author
item EWY, ROBERT
item PORTIS JR, ARCHIE

Submitted to: American Society of Plant Physiologists Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 8/6/1999
Publication Date: N/A
Citation: Ewy, R.G., Portis Jr, A.R. 1999. Altered atp-binding and atpase activity in rubisco activase mutants. American Society of Plant Physiologists Meeting: p.169

Interpretive Summary:

Technical Abstract: Rubisco activase (RA) is an ATP-dependent enzyme which removes sugar phosphate inhibitors from Rubisco, In Arabidopsis thaliana RA is present as both 43- and 46-kD polypetides which contain a conserved ATP-binding motif, the P-look. While the 43kD form of RA exhibits ATPase activity under phy7siological ATP/ADP ratios in vitro, the 46kD protein exhibits little ATPase activity under the same conditions. To better understand the mechanism of action of RA, we have characterized ATP-binding with the 43kD form of the enzyme in recombinant proteins with single-site mutations in the P-loop region. ATP-binding to the recombinant Q111D, Q111E, Q111S, and wt form of the 43kD RA was studied in vitro using intrinsic, 1,8-ANS, and TNP-ATP fluorescence. For each protein, the three methods usually gave different apparent K**ms for ATP-binding. These disparities probably reflect the dissimilar molecular interactions unique to each method. However, the Q111D recombinant protein had the least affinity for ATP than any of the other isozymes as shown by all three measurements. Despite the decreased affinity for ATP, the Q111D recombinant protein exhibited greater ATPase activity in vitro under physiological ATP/ADP ratios than either the rwt 43kD, Q111D or Q111S form of the enzyme. The Q111S recombinant protein showed much lower ATPase activity at physiological ATP/ADP ratios than any of the other enzymes tested. The results will be discussed with respect to the mutant proteins' ability to activate Rubisco. This research was sponsored, in part by DOE grant 97ER20268.