Identification of critical arginine residues in the functioning of Rubisco activase.
Rubisco activase is a member of the AAA(+) family in which arginines located in the Box VII and Sensor 2 domains are a recurrent feature and typically contribute to ATP-binding/hydrolysis or an inter-subunit interface. Replacement of R241 or R244 in Box VII or R294 or R296 in Sensor 2 with alanine in tobacco activase did not greatly alter the binding of ATP or ADP. However, ATP hydrolysis was minimal (R241A and R244A) or greatly diminished (R296A) and none of these mutants were able to activate Rubisco. R241, R244 and R296 were also required for nucleotide-dependent conformational changes detected by intrinsic fluorescence and limited proteolysis. ATP-induced oligomerization, monitored by gel filtration, was not observed with the wild type and mutant tobacco activases in contrast to spinach activase and a R239A mutant (corresponding to R244A in tobacco). Thus, there is not a strict correlation of oligomerization with ATP hydrolysis and intrinsic fluorescence.
Li
Cishan
C
Department of Plant Biology, University of Illinois, 190 ERML, 1201 W Gregory Ave., Urbana IL 61801, USA.
Wang
Dafu
D
Portis
Archie R
AR
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D004789:Enzyme Activation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism*; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology
D010940:Plant Proteins; D011994:Recombinant Proteins; C054765:rca protein, plant; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D012273:Ribulose-Bisphosphate Carboxylase