Reductive half-reaction of thioredoxin reductase from Escherichia coli.
Thioredoxin reductase is a homodimeric flavoenzyme containing a flavin adenine dinucleotide (FAD) and a redox-active disulfide in each subunit. Structural work on the enzyme from Escherichia coli suggests that thioredoxin reductase exists in two conformations, both of which are necessary for catalysis [Waksman, G., Krishna, T. S. R., Williams, C. H., Jr., & Kuriyan, J. (1994) J. Mol. Biol. 236, 800-816]. These factors make it likely that the mechanism of this enzyme is complex. The rapid reaction of enzyme with nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) (the reductive half-reaction), proceeds in three phases. The first phase represents the formation of an NADPH-FAD charge transfer complex. The second phase involves FAD reduction, with loss of the NADPH-FAD charge transfer band. The third phase shows a slower decrease in absorbance at 456 nm and the formation of a reduced flavin-NADP+ charge transfer band. These and other results indicate that NADP+ and NADPH compete for the single binding site on oxidized and fully reduced enzyme and that NADP+ release does not limit the third phase of reduction. Experiments that include examination of the reductive half-reactions of active-site mutants, having the active-site disulfide removed by mutating one or both of the active-site cysteines, indicate that the third phase does not represent reduction by a second equivalent of NADPH. Comparison of the rate constants and temperature dependence of the reductive half-reaction with those of turnover show that the reductive half-reaction is not solely rate-limiting in catalysis. The results suggest that wild type and each altered enzyme exists in a unique equilibrium of conformers. It is proposed that the third phase of the reductive half-reaction represents a flavin reduction event largely limited by the conformational change proposed in the structural work.
Lennon
B W
BW
Department of Biological Chemistry, University of Michigan, and Department of Veterans Affairs Medical Center, Ann Arbor, Michigan 48105, USA.
Williams
C H
CH
D004926:Escherichia coli / Q000201:enzymology*; D005415:Flavins / Q000737:chemistry; D009249:NADP / Q000737:chemistry; D010084:Oxidation-Reduction; D013816:Thermodynamics; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry*
D005415:Flavins; D009249:NADP; D013880:Thioredoxin-Disulfide Reductase