title: Effect of pyridine nucleotide on the oxidative half-reaction of Escherichia coli thioredoxin reductase.
abstract: The kinetics of the oxidative half-reaction between reduced thioredoxin reductase and oxidized thioredoxin measured in the presence and absence of pyridine nucleotide show a significant difference in the rates of the main phase of oxidation. When 1 equiv of NADPH is used to partially reduce the enzyme at pH 7.0 or 7.6, the observed rate of the catalytically competent phase of oxidation is essentially equal to kcat at that pH. This is about 50% of the rate of oxidation observed with enzyme fully reduced or partially reduced by the xanthine/xanthine oxidase system or by dithionite. Through the use of the nonreducible analog 3-aminopyridine adenine dinucleotide phosphate we have shown that this decrease in observed rate of oxidation is linked to the concentration of pyridine nucleotide present. This suggests 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. This is the case even when substoichiometric quantities of 3-aminopyridine adenine dinucleotide phosphate are present, which predicts that the binding to reduced enzyme is very tight. It is clear that the presence of 1 equiv of NADP+ is sufficient to cause the observed rate for the catalytically competent phase of oxidation to decrease to kcat. Thus, there is compelling evidence for a ternary complex mechanism for thioredoxin reductase.
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor.
authors.forename: B W
authors.initials: BW
authors.lastname: Lennon
authors.forename: C H
authors.initials: CH
authors.lastname: Williams
mesh_terms: D004926:Escherichia coli / Q000201:enzymology*; D006207:Half-Life; D007700:Kinetics; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism*
chemical_list: D009249:NADP; D013880:Thioredoxin-Disulfide Reductase