INVOLVEMENT OF 2 ASPARTATE RESIDUES OF RUBISCO ACTIVASE IN COORDINATION OF THE ATP GAMMA-PHOSPHATE AND SUBUNIT COOPERATIVITY

Aspartate residues are involved in coordination of the nucleotide-meta l of several nucleotide triphosphatases. To examine interactions betwe en 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 t he presence of Mg2+, the affinity for ATP of D231R was further increas ed, but was reduced with mutations at D174. Although all mutants bound ATP, only D174E aggregated in response to ATP/Mg2+ and retained parti al ATPase and Rubisco activation activities. In mixing experiments, th e catalytically competent D174E stimulated wild-type ATPase activity, whereas the mutants lacking ATPase activity were inhibitory to wild-ty pe enzyme and prevented aggregation. These results are consistent with a mechanism for activase that involves ATP-binding, subunit aggregati on and ATP hydrolysis as sequential steps in the catalytic mechanism. The results also indicated that precise coordination of the gamma-phos phate is required for aggregation and depends on D174 and D231. To acc ount for the pronounced cooperativity of Rubisco activase subunits, we suggest that coordination of the ATP gamma-phosphate may involve part icipation of residues from adjacent subunits.