Site-directed mutagenesis of a reactive lysyl residue (Lys-247) of Rubisco activase

Arch Biochem Biophys. 1994 Oct;314(1):178-85. doi: 10.1006/abbi.1994.1427.


Chemical modification of tobacco leaf ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase with water-soluble N-hydroxysuccinimide esters identified Lys-247 as a particularly reactive residue necessary for maximal catalytic activity [M.E. Salvucci (1993) Plant Physiol. 103, 501-508]. To further explore the role of Lys-247 in catalysis, this species-invariant residue of Rubisco activase was changed to Arg, Cys, and Gln by mutagenesis of a cDNA clone of the mature form of the tobacco enzyme. Analysis of the purified recombinant proteins showed that all three point mutations reduced the rate of ATP hydrolysis to 2 to 3% of the wild-type enzyme and completely abolished the ability of Rubisco activase to promote activation of decarbamylated Rubisco. Replacement of Lys-247 with Arg, Cys, or Gln had a comparatively minor effect on ATP binding, but eliminated the increase in ATPase-specific activity that normally occurs with increasing concentrations of Rubisco activase protein. In mixing experiments, the K247R mutant enzyme inhibited Rubisco activation by wild-type Rubisco activase, indicating that interactions between Rubisco and Rubisco activase were disrupted by even the most conservative of the substitutions. Chemical elaboration of the K247C mutant by treatment with 2-bromoethylamine converted 39% of the thiols at position 247 to the aminoethyl derivative, but failed to improve the catalytic performance of the mutant enzyme. Our results indicate that the requirement for a lysyl residue at position 247 of Rubisco activase is very stringent, consistent with its proposed role in coordinating precise interactions with gamma-phosphate of ATP.

MeSH terms

  • Adenosine Triphosphatases / metabolism
  • Adenosine Triphosphate / metabolism
  • Affinity Labels
  • Arginine
  • Cysteine
  • Electrophoresis, Polyacrylamide Gel
  • Glutamine
  • Isoelectric Focusing
  • Lysine / chemistry
  • Lysine / genetics*
  • Mutagenesis, Site-Directed*
  • Photochemistry
  • Plant Proteins*
  • Plants, Toxic
  • Point Mutation
  • Recombinant Proteins
  • Ribulose-Bisphosphate Carboxylase / chemistry
  • Ribulose-Bisphosphate Carboxylase / genetics*
  • Ribulose-Bisphosphate Carboxylase / metabolism
  • Structure-Activity Relationship
  • Tobacco / enzymology


  • Affinity Labels
  • Plant Proteins
  • Recombinant Proteins
  • rca protein, plant
  • Glutamine
  • Adenosine Triphosphate
  • Arginine
  • Adenosine Triphosphatases
  • Ribulose-Bisphosphate Carboxylase
  • Lysine
  • Cysteine