Effects of five-tryptophan mutations on structure, stability and function of Escherichia coli dihydrofolate reductase.
To elucidate the roles of tryptophan residues in the structure, stability, and function of Escherichia coli dihydrofolate reductase (DHFR), its five tryptophan residues were replaced by site-directed mutagenesis with leucine, phenylalanine or valine (W22F, W22L, W30L, W47L, W74F, W74L, W133F, and W133V). Far-ultraviolet circular dichroism (CD) spectra of these mutants reveal that exciton coupling between Trp47 and Trp74 strongly affects the peptide CD of wild-type DHFR, and that Trp133 also contributes appreciably. No additivity was observed in the contributions of individual tryptophan residues to the fluorescence spectrum of wild-type DHFR, Trp74 having a dominant effect. These single-tryptophan mutations induce large changes in the free energy of urea unfolding, which showed values of 1.79-7.14 kcal/mol, compared with the value for wild-type DHFR of 6.08 kcal/mol. Analysis of CD and fluorescence spectra suggests that thermal unfolding involves an intermediate with the native-like secondary structure, the disrupted Trp47-Trp74 exciton coupling, and the solvent-exposed Trp30 and Trp47 side chains. All the mutants except W22L (13%) retain more than 50% of the enzyme activity of wild-type DHFR. These results demonstrate that the five tryptophan residues of DHFR play important roles in its structure and stability but do not crucially affect its enzymatic function.
Ohmae
E
E
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526, Japan.
Sasaki
Y
Y
Gekko
K
K
D002942:Circular Dichroism; D004795:Enzyme Stability / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed / Q000235:genetics* / Q000502:physiology; D017510:Protein Folding; D013050:Spectrometry, Fluorescence / Q000379:methods; D013696:Temperature; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D014364:Tryptophan / Q000235:genetics* / Q000378:metabolism; D014508:Urea / Q000737:chemistry
D014364:Tryptophan; D014508:Urea; D013762:Tetrahydrofolate Dehydrogenase