G33D mutant thioredoxin primarily affects the kinetics of reaction with thioredoxin reductase. Probing the structure of the mutant protein.
Escherichia coli thioredoxin is a redox-active protein. A mutant protein with an aspartic acid substitution for the largely conserved glycine at position 33 (G33D) in the active site of thioredoxin has been generated to study the effects of a negatively charged residue in the active site of the protein. Despite the close proximity of the negative-charged Asp to the redox active cysteines, the effective concentration of the cysteines does not deviate significantly from that of the wild-type protein. The redox potential (E(o)') measured by the equilibrium between NADPH and the mutant thioredoxin is also close to that of the wild-type. Kinetic measurements of the reaction between thioredoxin and thioredoxin reductase show that G33D mutant and the wild-type proteins have identical kcat values. However, the Km for G33D mutant is approximately 10-fold higher than that for the wild-type protein. In vivo assay of the growth of E. coli strain carrying wild-type or G33D mutant thioredoxin on methionine sulfoxide indicates that the G33D mutant protein is a slower electron donor for methionine sulfoxide reductase. Structural stability of the oxidized protein is not altered by the G33D substitution, as illustrated by the same unfolding free energies studied by urea. The substitution does not show significant change of the near UV and far-UV circular dichroic (CD) and the fluorescence spectra for either the reduced or the oxidized protein. Therefore, the global structure of the G33D protein is not changed. However, the surface of the active site has been altered locally by G33D substitution, which accounts for the above kinetically poor behaviors. A model of G33D structure is constructed based on these studies.
Lin
T Y
TY
Institute of Biological Science and Technology, National Chiao Tung University, Taiwan, Republic of China. tylin@cc.nctu.edu.tw
D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics*; D002942:Circular Dichroism; D004579:Electron Transport; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D005998:Glycine / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry* / Q000378:metabolism; D013879:Thioredoxins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D001224:Aspartic Acid; D013879:Thioredoxins; D013880:Thioredoxin-Disulfide Reductase; D005998:Glycine