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Doctoral Dissertations
Date of Award
8-1997
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Major
Biomedical Sciences
Major Professor
Fred C. Hartman
Committee Members
Jorge Churchich, Margaret Terzaghi-Howe, Richard Mural
Abstract
Two chloroplastic thioredoxins, denoted Trx ƒ and Trx m, mediate photosynthetic light regulation of carbon dioxide assimilation via modulation of the redox status of certain target enzymes. The necessity for distinct chloroplastic thioredoxins has been viewed as reflective of their differential selectivities for these target enzymes. Trx ƒ has been reported to be selective for fructose-1,6-bisphosphatase (FBPase), phosphoribulokinase (PRK), sedoheptulose-1,7-bisphosphatase, and NADP-linked glyceraldehyde 3-phosphate dehydrogenase. Trx m, on the other hand, has appeared to be selective for NADP- dependent malate dehydrogenase (MDH) and chloroplastic glucose 6-phosphate dehydrogenase.
One objective of this project was to identify recognition and selectivity determinants of Trx ƒ. Amino acid sequence alignments and three-dimensional structures of E. coli Trx were used to identify residues of Trx ƒ that appear to be unique and accessible for interaction with target enzymes. These Trx ƒ residues were then replaced with those found at corresponding positions of Trx m, resulting in the following site-directed mutants: K58E, Q75D, N74D, the deletion mutants ΔAsn74 and ΔAsn77, T1051, and the double mutant V89I/T1051. Through kinetic analyses of the mutants constructed, Lys58, Asn74, Gln75, and Asn77 of Trx f were found to contribute to its interactions with FBPase and MDH and influence target enzyme selectivity. Val89 and Thr105 were also determined to be important for binding to FBPase. However, contrary to literature reports, wild-type Trx ƒ is superior to wild-type Trx m in the activation of both FBPase and MDH. Furthermore, the wild-type and mutant thioredoxins display similar efficiencies in the activation of PRK. Thus, the physiological basis for the two thioredoxins cannot be explained on the basis of differential target enzyme specificity as heretofore accepted.
A second objective of this project was to ascertain the feasibility of using fluorescence anisotropy to directly measure the binding affinities of the thioredoxins for their target enzymes. For this purpose, the wild-type chloroplastic thioredoxins were fluorescently labeled with isatoic anhydride. The affinity (Kd) of derivatized Trx ƒ for PRK was only twice as large as the apparent affinity value determined by kinetic analyses. In addition, the Kd values for derivatized Trx ƒ and m for PRK differed by only two-fold. d Derivatization, however, greatly impaired the ability of the thioredoxins to activate their targets. Thus, a residue involved in Trx-target enzyme interaction may have been modified.
Recommended Citation
Geck, Mary Kaye, "Characterization of the thioredoxin-modulated regulation of fructose-1, 6-bisphosphatase, NADP-dependent malate dehydrogenase, and phosphoribulokinase. " PhD diss., University of Tennessee, 1997.
https://trace.tennessee.edu/utk_graddiss/9499