Expression of the two isoforms of spinach ribulose 1,5-bisphosphate carboxylase activase and essentiality of the conserved lysine in the consensus nucleotide-binding domain.
The two isoforms of ribulose 1,2-bisphosphate carboxylase activase (Rbu-P2 carboxylase) from spinach (Spinacea oleracea L.) were individually purified from Escherichia coli transformed with expression vectors for the appropriate cDNAs. Both isoforms catalyzed activation of Rbu-P2 carboxylase (ribulose 1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39) and ATP hydrolysis. The kinetics of the two isoforms with respect to ATP concentration were different, in that the 45-kDa polypeptide exhibited a sigmoidal response while a rectangular response was observed with the 41-kDa isoform. These observations suggest that the additional domain at the C terminus of the 45-kDa isoform modulates the ATP regulation of activity. Lysine 169, at the putative ATP-binding site of the 41-kDa form of Rbu-P2 carboxylase activase, was changed to arginine, isoleucine, and threonine by directed mutagenesis. These mutations abolished Rbu-P2 carboxylase activase and ATPase activities, as well as the capability of the protein to bind ATP. These results confirm that lysine 169 is an essential residue.
Shen
J B
JB
Department of Agronomy, University of Illinois, Urbana-Champaign.
Orozco
E M
EM
Ogren
W L
WL
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship
D010940:Plant Proteins; C054765:rca protein, plant; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D012273:Ribulose-Bisphosphate Carboxylase; D008239:Lysine