Study of the reductive and oxidative half reactions of Escherichia coli thioredoxin reductase: Implications for catalysis.

Abstract

Thioredoxin reductase from E. coli is a dimeric flavoenzyme containing an FAD and a redox active disulfide in each subunit. Recent structural work indicates that thioredoxin reductase exists in two conformations, both of which are necessary for catalysis. These factors make it likely that the mechanism of this enzyme is quite complex. While steady state assay data have suggested a ping-pong mechanism, data are presented which indicate a ternary complex mechanism applies to this enzyme. The reductive half reactions of wild type thioredoxin reductase and four active site disulfide mutants with NADPH were studied. The results show that the reductive half reaction is not solely rate-limiting in turnover, and that disruption of the active site disulfide through mutation has significant effects on the reductive half reaction. These effects are not caused solely by the loss of the disulfide, but also by the nature of the residue(s) replacing the active site cysteine(s). It is proposed that of the three phases of reduction observed, the third phase represents the rate of the change between the two conformations proposed. Kinetic modeling of mechanisms based on this assumption show that the conformational change is probably not fast, and that such a model can produce three phases of reduction. The kinetics of the oxidative half reaction between reduced thioredoxin reductase and oxidized thioredoxin measured in the presence and absence of pyridine nucleotides show a significant difference in the rates of the main phase of oxidation. Through the use of the non-reducible analog 3-aminopyridine adenine dinucleotide phosphate it was shown that this decrease in the observed rate of oxidation is linked to the concentration of pyridine nucleotide present. This indicates that the complexation of pyridine nucleotides with reduced thioredoxin reductase is able to effect a change in the rate-limiting steps of the oxidation of the enzyme by thioredoxin. Enzyme-monitored turnover experiments show that the flavin in thioredoxin reductase is essentially fully reduced during turnover, indicating that the catalytic cycle involves two electron-reduced and four electron-reduced enzyme forms. Spectra taken during turnover indicate that turnover occurs with NADPH bound. Thus, there is compelling evidence for a ternary complex mechanism for thioredoxin reductase.