It has been proposed that an acid-base catalyst facilitates the reduction of thioredoxin by thioredoxin reductase from Escherichia coli [O'Donnell, M. E., & Williams, C. H. Jr. (1983) J. Biol. Chem. 252, 13795-13805]. The X-ray crystal structure reveals two groups which could potentially fulfill this role: His245 and Asp139. Using site-directed mutagenesis, His245 was changed to asparagine (H245N) and alanine (H245A) and Asp139 was changed to glutamate (D139E), asparagine (D139N), and leucine (D139L). Steady-state kinetic analysis of the His245 mutants gave turnover numbers and Km values similar to those of wild-type thioredoxin reductase. All three Asp139 mutants were altered in their overall kinetic properties: D139E had 38% of wild-type activity, D139N had 1.5%, and D139L had no measureable activity. Rate constants for the NADPH to 3-acetylpyridine adenine dinucleotide phosphate transhydrogenase activity were similar for all of the Asp139 and His245 mutants and wild-type thioredoxin reductase. Stopped-flow kinetic measurements of the reductase half-reaction of H245A and H245N gave rate constants that were up to 2-fold faster than those found for wild-type thioredoxin reductase, while all of the Asp139 mutants had rate constants comparable to those of wild-type. To further examine the causes of the low overall activity of D139N, the oxidative half-reaction was measured. The reoxidation of reduced D139N mixed with oxidized thioredoxin occurred at a very slow rate constant of 0.23 s-1-about 1% that of wild-type enzyme. We suggest that Asp139 is the active-site acid catalyst which functions to protonate the thiolate anion of reduced thioredoxin.(ABSTRACT TRUNCATED AT 250 WORDS)