POTENTIAL ACTIVE-SITE BASE OF THIOREDOXIN REDUCTASE FROM ESCHERICHIA-COLI - EXAMINATION OF HISTIDINE(245) AND ASPARTATE(139) BY SITE-DIRECTED MUTAGENESIS

It has been proposed that an acid-base catalyst facilitates the reduction of thioredoxin by thioredoxin reductase from Escherichia coli [O'Donnell, M. E., and 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: His(245) and Asp(139), Using site-directed mutagenesis, His(245) was changed to asparagine (H245N) and alanine (H245A) and Asp(139) was changed to glutamate (D139E), asparagine (D139N), and leucine (D139L). Steady-state kinetic analysis of the His(245) mutants gave turnover numbers and K-m values similar to those of wild-type thioredoxin reductase. All three Asp(139) mutants were altered in their overall kinetic properties: D139E had 38% of wild-type activity, D139N had 1.5%, and D139L had no measurable activity. Rate constants for the NADPH to 3-acetylpyridine adenine dinucleotide phosphate transhydrogenase activity were similar for all of the Asp(139) and His(245) 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 Asp(139) 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 Asp(139) is the active-site acid catalyst which functions to protonate the thiolate anion of reduced thioredoxin. Thus, the reductive half-reaction is not affected in mutants of Asp(139); only the oxidative half-reaction is slowed, consistent with the proposed function of this residue as an acid-base catalyst.