Site-directed mutagenesis of a reactive lysyl residue (Lys-247) of Rubisco activase.
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.
Salvucci
M E
ME
U.S. Department of Agriculture, University of Kentucky, Lexington 40546-0076.
Klein
R R
RR
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels; D001120:Arginine; D003545:Cysteine; D004591:Electrophoresis, Polyacrylamide Gel; D005973:Glutamine; D007525:Isoelectric Focusing; D008239:Lysine / Q000737:chemistry / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D010940:Plant Proteins; D010947:Plants, Toxic; D017354:Point Mutation; D011994:Recombinant Proteins; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014026:Nicotiana / Q000201:enzymology
D000345:Affinity Labels; D010940:Plant Proteins; D011994:Recombinant Proteins; C054765:rca protein, plant; D005973:Glutamine; D000255:Adenosine Triphosphate; D001120:Arginine; D000251:Adenosine Triphosphatases; D012273:Ribulose-Bisphosphate Carboxylase; D008239:Lysine; D003545:Cysteine