Kinetic and Mutational Analyses of the Regulation of Phosphoribulokinase by Thioredoxins

• Published in: The Journal of biological chemistry Vol. 275; no. 24; pp. 18034 - 18039
• Main Authors: Geck, Mary K., Hartman, Fred C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 16.06.2000; American Society for Biochemistry and Molecular Biology
• Subjects: BASIC BIOLOGICAL SCIENCES; BIOLOGY; CHEMISTRY; Chloroplast Thioredoxins; Chloroplasts - chemistry; Dithiothreitol - pharmacology; DNA Mutational Analysis; Enzyme Activation; Glutathione - metabolism; KINETICS; Molecular Weight; Mutagenesis, Site-Directed; Phosphotransferases (Alcohol Group Acceptor) - metabolism; REGULATIONS; Thioredoxins - genetics; Thioredoxins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M001936200

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 Trxm to be more efficient than Trx f because of its 40% higher Vmax but similarS0.5. 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 Trxm counterparts or deleted. These changes generally resulted in Vmax 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.