The molecular pathway for the regulation of phosphoribulokinase by thioredoxin f.
Phosphoribulokinase (PRK) is one of several plant enzymes that is regulated by thiol-disulfide exchange as mediated by thioredoxin, which contains spatially vicinal, redox-active cysteinyl residues. In an earlier study (Brandes, H. K., Larimer, F. W., Geck, M. K., Stringer, C. D., Schürmann, P., and Hartman, F. C. (1993) J. Biol. Chem. 268, 18411-18414), our laboratory identified Cys-46 of thioredoxin f (Trx), as opposed to the other candidate Cys-49, as the primary nucleophile that attacks the disulfide of target proteins. The goal of the present study was to identify which of the two redox-active cysteinyl residues of PRK (Cys-16 or Cys-55) is paired with Cys-46 of Trx in the interprotein disulfide intermediate of the overall oxidation-reduction pathway. Incubation of a mixture of the C16S mutant of PRK and the C49S mutant of Trx with Cu2+ results in covalent complex formation as detected by SDS-polyacrylamide gel electrophoresis. Complexation is fully reversible by dithiothreitol and is retarded by ligands for PRK. Under the same conditions, Cu2+ induces very little complex formation between the following pairs of mutants: C16S PRK/C46S Trx, C55S PRK/C49S Trx, and C55S PRK/C46S Trx. When either 5-thio-2-nitrobenzoate-derivatized C16S or C55S PRK, as mimics of the oxidized (disulfide) form of the enzyme, is mixed with C49S Trx, stable covalent complex formation occurs only with the C16S PRK. Thus, two independent approaches identify Cys-55 of PRK in the intermolecular disulfide pairing with Trx.
Brandes
H K
HK
Protein Engineering Program, Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA.
Larimer
F W
FW
Hartman
F C
FC
D001665:Binding Sites; D054479:Chloroplast Thioredoxins; D003545:Cysteine; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D017354:Point Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013879:Thioredoxins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D054479:Chloroplast Thioredoxins; D004220:Disulfides; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D013879:Thioredoxins; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase; D003545:Cysteine