Kinetic and mutational analyses of the regulation of phosphoribulokinase by thioredoxins.
Despite little supportive data, differential target protein susceptibility to redox regulation by thioredoxin (Trx) f and Trx m has been invoked to account for two distinct Trxs in chloroplasts. However, this postulate has not been rigorously tested with phosphoribulokinase (PRK), a fulcrum for redox regulation of the Calvin cycle. Prerequisite to Trx studies, the activation of spinach PRK by dithiothreitol, 2-mercaptoethanol, and glutathione was examined. Contrary to prior reports, each activated PRK, but only dithiothreitol supported Trx-dependent activation. Comparative kinetics of activation of PRK showed Trx m to be more efficient than Trx f because of its 40% higher V(max) but similar S(0.5). Activations were insensitive to ribulosebisphosphate carboxylase, which may complex with PRK in vivo. To probe the basis for superiority of Trx m, we characterized site-directed mutants of Trx f, in which unique residues in conserved regions were replaced with Trx m counterparts or deleted. These changes generally resulted in V(max) enhancements, the largest (6-fold) of which occurred with T105I, reflective of substitution in a hydrophobic region that opposes the active site. Inclusive of the present study, activation kinetics of several different Trx-regulated enzymes indicate redundancy in the functions of the chloroplastic Trxs.
Geck
M K
MK
University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences and the Protein Engineering Program, Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA.
Hartman
F C
FC
D054479:Chloroplast Thioredoxins; D002736:Chloroplasts / Q000737:chemistry; D004252:DNA Mutational Analysis; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation; D005978:Glutathione / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D013879:Thioredoxins / Q000235:genetics* / Q000378:metabolism*
D054479:Chloroplast Thioredoxins; D013879:Thioredoxins; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase; D005978:Glutathione; D004229:Dithiothreitol