ACTIVATION OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE (RUBISCO) INVOLVES RUBISCO ACTIVASE TRP16/

The role of the N-terminal region of tobacco Rubisco activase in ATP h ydrolysis and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco ) activation was examined by construction of mutant proteins. Deletion of the first 50 amino acids of Rubisco activase almost completely eli minated the ability to activate Rubisco, without changing the ATP-hydr olyzing and self-associating properties of the enzyme. Thus, the N-ter minus of Rubisco activase is distinct from the ATP-hydrolyzing domain and is required for Rubisco activation, Directed mutagenesis of the sp ecies-invariant tryptophan residue at position 16 inhibited Rubisco ac tivation but not the binding or hydrolysis of ATP. The ability to acti vate Rubisco was less severely inhibited when Trp was replaced by a Ty r or Phe than by an Ala or Cys, indicating that an aromatic residue at position 16 and particularly a Trp is required for proper activation of Rubisco. Fluorescence quenching of the 7-nitrobenz-2-oxa-1,3-diazol e-modified W16C mutant upon addition of nucleotide suggested that posi tion 16 becomes more solvent accessible in response to nucleotide bind ing, However, changes in the intrinsic fluorescence of truncated and T rp16 mutants upon addition of ATP were similar to those of the wild ty pe, evidence that Trp16 is not the residue reporting the conformationa l change that accompanies subunit association.