Activation of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) involves Rubisco activase Trp16.
The role of the N-terminal region of tobacco Rubisco activase in ATP hydrolysis 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 eliminated the ability to activate Rubisco, without changing the ATP-hydrolyzing and self-associating properties of the enzyme. Thus, the N-terminus of Rubisco activase is distinct from the ATP-hydrolyzing domain and is required for Rubisco activation. Directed mutagenesis of the species-invariant tryptophan residue at position 16 inhibited Rubisco activation but not the binding or hydrolysis of ATP. The ability to activate Rubisco was less severely inhibited when Trp was replaced by a Tyr 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-diazole-modified W16C mutant upon addition of nucleotide suggested that position 16 becomes more solvent accessible in response to nucleotide binding. However, changes in the intrinsic fluorescence of truncated and Trp16 mutants upon addition of ATP were similar to those of the wild type, evidence that Trp16 is not the residue reporting the conformational change that accompanies subunit association.
van de Loo
F J
FJ
United States Department of Agriculture, Agricultural Research Service, Western Cotton Research Laboratory, Phoenix, Arizona 85040-8830, USA.
Salvucci
M E
ME
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D004789:Enzyme Activation; D005470:Fluorometry; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins; D010947:Plants, Toxic; D011994:Recombinant Proteins / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000201:enzymology; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
D010940:Plant Proteins; D011994:Recombinant Proteins; C054765:rca protein, plant; D014364:Tryptophan; D000255:Adenosine Triphosphate; D012273:Ribulose-Bisphosphate Carboxylase