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.