Rapeseed chloroplast thioredoxin-m. Modulation of the affinity for target proteins.
The stroma of higher plant chloroplasts contains two thioredoxins (Trx) with different specificity for the reduction of protein disulfide bonds. Based upon electrostatic features of domains that participate in the thiol/disulfide exchange, we prepared mutants of rapeseed Trx-m bearing opposite charges at a single position and subsequently analyzed their action on the activation of rapeseed chloroplast fructose 1,6-phosphate (CFBPase). The replacement of Pro-35 with lysine and glutamic residues enhanced and impaired, respectively, the stimulation of CFBPase relative to the wild-type and the P35A mutant. Furthermore, the shielding of electrostatic interactions with high concentrations of KCl greatly increased and concurrently made indistinguishable the affinity of all variants for CFBPase. The capacity to stimulate the enzyme activity likewise was enhanced concertedly by fructose-1,6-bisphosphate and Ca(2+) but, at variance with the action of KCl, remained sensitive to charges in the side chain of mutants. These results were consistent with a mechanism in which intermolecular electrostatic interactions and intramolecular non-covalent interactions control the formation of the non-covalent complex between reduced Trx and oxidized CFBPase and, in so doing, modulate the thiol/disulfide exchange.
Duek
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Instituto Leloir, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Patricias Argentinas 435, 1405, Buenos Aires, Argentina. pduek@iib.uba.ar
Wolosiuk
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D019943:Amino Acid Substitution; D001937:Brassica / Q000201:enzymology* / Q000235:genetics; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D054479:Chloroplast Thioredoxins; D002736:Chloroplasts / Q000201:enzymology*; D003198:Computer Simulation; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D006597:Fructose-Bisphosphatase / Q000378:metabolism*; D005635:Fructosediphosphates / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D017510:Protein Folding; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000502:physiology; D013816:Thermodynamics; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D054479:Chloroplast Thioredoxins; D005635:Fructosediphosphates; D013879:Thioredoxins; D011189:Potassium Chloride; D006597:Fructose-Bisphosphatase; C029063:fructose-1,6-diphosphate; D002118:Calcium