Authors: Ohmae, Eiji¹; Fukumizu, Yukari¹; Iwakura, Masahiro²; Gekko, Kunihiko¹

Source: The Journal of Biochemistry, Volume 137, Number 5, May 2005 , pp. 643-652(10)

Publisher: Oxford University Press

Abstract:

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, G°<inf>u</inf>, increased with increases in the side-chain hydrophobicity in the range 2.96–6.40 kcal·mol^–1, which includes the value for the wild-type enzyme (6.08 kcal·mol^–1). The steady-state kinetic parameters, K <inf>m</inf> and k <inf>cat</inf>, also increased with increases in the side-chain hydrophobicity, with the M42W mutant showing the largest increases in K <inf>m</inf> (35-fold) and k <inf>cat</inf> (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.

Document Type: Research article

DOI: http://dx.doi.org/10.1093/jb/mvi079

Affiliations: 1: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526; and 2: National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566

Publication date: 2005-05-01

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