Involvement of two aspartate residues of Rubisco activase in coordination of the ATP gamma-phosphate and subunit cooperativity.
Aspartate residues are involved in coordination of the nucleotide-metal of several nucleotide triphosphatases. To examine interactions between Rubisco activase and ATP, site-directed mutations were made at two species-invariant aspartate residues, D174 and D231. In the absence of the magnesium cofactor, the mutant proteins D231R, D174Q, and D174A, but not D174E, bound ATP with higher affinity than did wild-type. In the presence of Mg2+, the affinity for ATP of D231R was further increased, but was reduced with mutations at D174. Although all mutants bound ATP, only D174E aggregated in response to ATP/Mg2+ and retained partial ATPase and Rubisco activation activities. In mixing experiments, the catalytically competent D174E stimulated wild-type ATPase activity, whereas the mutants lacking ATPase activity were inhibitory to wild-type enzyme and prevented aggregation. These results are consistent with a mechanism for activase that involves ATP-binding, subunit aggregation and ATP hydrolysis as sequential steps in the catalytic mechanism. The results also indicated that precise coordination of the gamma-phosphate is required for aggregation and depends on D174 and D231. To account for the pronounced cooperativity of Rubisco activase subunits, we suggest that coordination of the ATP gamma-phosphate may involve participation of residues from adjacent subunits.
van de Loo
F J
FJ
Agricultural Research Service, Western Cotton Research Laboratory, United States Department of Agriculture, 4135 East Broadway Road, Phoenix, Arizona 85040-8830, USA. F.vandeLoo@pi.csiro.au
Salvucci
M E
ME
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D010947:Plants, Toxic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology
D010940:Plant Proteins; D011994:Recombinant Proteins; C054765:rca protein, plant; D001224:Aspartic Acid; D000255:Adenosine Triphosphate; D008274:Magnesium