Alteration of the adenine nucleotide response and increased Rubisco activation activity of Arabidopsis rubisco activase by site-directed mutagenesis.
Arabidopsis Rubisco was activated in vitro at rates 2- to 3-fold greater by recombinant Arabidopsis 43-kD Rubisco activase with the amino acid replacements Q111E and Q111D in a phosphate-binding loop, G-G-K-G-Q-G-K-S. However, these two mutant enzymes had only slightly greater rates of ATP hydrolysis. Activities of the Q111D enzyme were much less sensitive and those of Q111E were somewhat less sensitive to inhibition by ADP. Both mutant enzymes exhibited higher Rubisco activation activities over the physiological range of ADP to ATP ratios. Enzymes with non-polar, polar, and basic residues substituted at position Gln-111 exhibited rates of Rubisco activation less than the wild-type enzyme. Estimates of the relative affinity of the wild type and the Q111D, Q111E, and Q111S enzymes for adenosine nucleotides by a variety of methods revealed that the nucleotide affinities were the most diminished in the Q111D enzyme. The temperature stability of the Q111D and Q111E enzymes did not differ markedly from that of the 43-kD recombinant wild-type enzyme, which is somewhat thermolabile. The Q111D and Q111E enzymes, expressed in planta, may provide a means to better define the role of the ADP to ATP ratio in the regulation of Rubisco activation and photosynthesis rate.
Kallis
R P
RP
Department of Plant Biology, University of Illinois, Urbana, Illinois 61801, USA.
Ewy
R G
RG
Portis
A R
AR
D000244:Adenosine Diphosphate / Q000378:metabolism* / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D005453:Fluorescence; D006358:Hot Temperature; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism*
D010940:Plant Proteins; C054765:rca protein, plant; D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases; D012273:Ribulose-Bisphosphate Carboxylase