Potential active-site base of thioredoxin reductase from Escherichia coli: examination of histidine245 and aspartate139 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., & 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)
Mulrooney
S B
SB
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
Williams
C H
CH
D001224:Aspartic Acid; D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006639:Histidine; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001224:Aspartic Acid; D006639:Histidine; D009249:NADP; D013880:Thioredoxin-Disulfide Reductase