Abstract
The thioredoxin superfamily consists of enzymes that catalyze the reduction, formation, and isomerization of disulfide bonds and exert their activity through a redox active disulfide in a Cys-Xaa(1)-Xaa(2)-Cys motif. The individual members of the family differ strongly in their intrinsic redox potentials. However, the role of the different redox potentials for the in vivo function of these enzymes is essentially unknown. To address the question of in vivo importance of redox potential for the most reducing member of the enzyme family, thioredoxin, we have employed a set of active site variants of thioredoxin with increased redox potentials (-270 to -195 mV) for functional studies in the cytoplasm of Escherichia coli. The variants proved to be efficient substrates of thioredoxin reductase, providing a basis for an in vivo characterization of NADPH-dependent reductive processes catalyzed by the thioredoxin variants. The reduction of sulfate and methionine sulfoxide, as well as the isomerization of periplasmic disulfide bonds by DsbC, which all depend on thioredoxin as catalyst in the E. coli cytoplasm, proved to correlate well with the intrinsic redox potentials of the variants in complementation assays. The same correlation could be established in vitro by using the thioredoxin-catalyzed reduction of lipoic acid by NADPH as a model reaction. We propose that the rate of direct reduction of substrates by thioredoxin, which largely depends on the redox potential of thioredoxin, is the most important parameter for the in vivo function of thioredoxin, as recycling of reduced thioredoxin through NADPH and thioredoxin reductase is not rate-limiting for its catalytic cycle.
Highlights
The thioredoxin superfamily consists of enzymes that catalyze the reduction, formation, and isomerization of disulfide bonds and exert their activity through a redox active disulfide in a Cys-Xaa1-Xaa2-Cys motif
We propose that the rate of direct reduction of substrates by thioredoxin, which largely depends on the redox potential of thioredoxin, is the most important parameter for the in vivo function of thioredoxin, as recycling of reduced thioredoxin through NADPH and thioredoxin reductase is not rate-limiting for its catalytic cycle
As a prerequisite for an in vivo characterization of the active site variants of E. coli thioredoxin listed in Table I, it had to be proven that the variants are still efficient substrates of thioredoxin reductase (TR)
Summary
Materials—DE52 and CM52 cellulose were from Whatman, the Superdex 200 HiLoad 26/60 column and the PD10 columns were from Amersham Pharmacia Biotech, and 2Ј,5Ј-ADP-agarose was from Sigma. In addition to the increase in optical density at 650 nm, the reaction at pH 8.0 was followed by the consumption of NADPH (⑀340 nm ϭ 6200 MϪ1 cmϪ1, 0.1-cm quartz cuvettes) and by HPLC analysis of the insulin reduction. For the latter analysis aliquots were removed from the reaction at different time points, the reactions were quenched with formic acid (20% (w/v) final concentration), and the insulin reaction products were separated by reversed-phase HPLC at 55 °C on a Vydac 218TP54 column using a linear gradient from 30 to 60% acetonitrile in 0.1% trifluoroacetic acid.
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