Effects of mutation at methionine-42 of Escherichia coli dihydrofolate reductase on stability and function: implication of hydrophobic interactions.
Methionine-42, distal to the active site of Escherichia coli dihydrofolate reductase, was substituted by site-directed mutagenesis with 14 amino acids (Ala, Cys, Glu, Gln, Gly, His, Ile, Leu, Pro, Ser, Thr, Trp, Tyr, and Val) to elucidate its role in the stability and function of this enzyme. Far-ultraviolet circular dichroism spectra of these mutants showed a distinctive negative peak at around 230 nm beside 220 nm, depending on the hydrophobicity of the amino acids introduced. The fluorescence intensity also increased in an order similar to that of the amino acids. These spectroscopic data suggest that the mutations do not affect the secondary structure, but strongly perturb the exciton coupling between Trp47 and Trp74. The free energy of urea unfolding, deltaG(o)u, increased with increases in the side-chain hydrophobicity in the range 2.96-6.40 kcal x mol(-1), which includes the value for the wild-type enzyme (6.08 kcal x mol(-1)). The steady-state kinetic parameters, Km and kcat, also increased with increases in the side-chain hydrophobicity, with the M42W mutant showing the largest increases in Km (35-fold) and kcat (4.3-fold) compared with the wild-type enzyme. These results demonstrate that site 42 distal to the active site plays an important role in the stability and function of this enzyme, and that the main effect of the mutations is to modify of hydrophobic interactions with the residues surrounding this position.
Ohmae
Eiji
E
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526.
Fukumizu
Yukari
Y
Iwakura
Masahiro
M
Gekko
Kunihiko
K
D002942:Circular Dichroism; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D006358:Hot Temperature; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008715:Methionine / Q000235:genetics*; D008958:Models, Molecular; D017354:Point Mutation / Q000235:genetics*; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism*
D008715:Methionine; D013762:Tetrahydrofolate Dehydrogenase