title: Enzyme-monitored turnover of Escherichia coli thioredoxin reductase: insights for catalysis.
abstract: Thioredoxin reductase from Escherichia coli is a member of the pyridine nucleotide-disulfide oxidoreductase family, and contains one FAD and one redox-active disulfide per subunit. It is known that two other well-studied members of this family, lipoamide dehydrogenase and glutathione reductase, cycle between the two electron-reduced and fully oxidized forms in catalysis. Enzyme-monitored turnover shows that the spectrum of thioredoxin reductase during turnover represents fully reduced flavin with NADP(H) bound. Whether the pyridine nucleotide bound is NADPH or NADP+ is dependent on the concentration of each species, i.e., how far turnover has progressed. It is also shown that the midpoint potentials of this enzyme are increased through the differential binding of NADP+ to the oxidized and reduced form of the enzyme. When combined with other kinetic and oxidation/reduction studies of this enzyme, these results indicate that thioredoxin reductase cycles between the four-electron-reduced and two-electron-reduced forms in catalysis, and that it does so with pyridine nucleotide bound. These results clarify the mechanism of thioredoxin reductase in relation to the known structure the enzyme, and provide support for earlier work in which we proposed that this enzyme utilizes a ternary complex mechanism in catalysis.
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, 48105, USA.
authors.forename: B W
authors.initials: BW
authors.lastname: Lennon
authors.forename: C H
authors.initials: CH
authors.lastname: Williams
mesh_terms: D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry; D013379:Substrate Specificity; D013880:Thioredoxin-Disulfide Reductase / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D009249:NADP; D013880:Thioredoxin-Disulfide Reductase