Effects of mutation at methionine-42 of Escherichia coli dihydrofolate reductase on stability and function: implication of hydrophobic interactions.

Effects of mutation at methionine-42 of Escherichia coli dihydrofolate reductase on stability and function: implication of hydrophobic interactions. Research Abstract Details 

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Effects of mutation at methionine-42 of Escherichia coli dihydrofolate reductase on stability and function: implication of hydrophobic interactions. Abstract Text:

Eiji Ohmae,Yukari Fukumizu,Masahiro Iwakura,Kunihiko Gekko,

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.

Effects of mutation at methionine-42 of Escherichia coli dihydrofolate reductase on stability and function: implication of hydrophobic interactions. Publishing Authors By Initials

E Ohmae,Y Fukumizu,M Iwakura,K Gekko,

For similar enzymes and coenzymes: enzymes: oxidoreductases: oxidoreductases acting on ch-nh group donors: tetrahydrofolate dehydrogenase research abstracts see: enzymes and coenzymes: enzymes: oxidoreductases: oxidoreductases acting on ch-nh group donors: tetrahydrofolate dehydrogenase research

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Effects of mutation at methionine-42 of Escherichia coli dihydrofolate reductase on stability and function: implication of hydrophobic interactions. Journal Published:

PUBLICATION TYPE: Journal Article

Journal: Journal of biochemistry

VOLUME: 137

Page Numbers: 643-52

Journal Abbreviation: J. Biochem.

ISSN: 0021-924X

DAY: 19

MONTH: May

YEAR: 2005

Effects of mutation at methionine-42 of Escherichia coli dihydrofolate reductase on stability and function: implication of hydrophobic interactions. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 376600

Effects of mutation at methionine-42 of Escherichia coli dihydrofolate reductase on stability and function: implication of hydrophobic interactions. Keywords Mesh Terms:

KEYWORDS: Tetrahydrofolate Dehydrogenase

MESH TERMS: metabolism

Chemical & Substance for Abstract: Effects of mutation at methionine-42 of Escherichia coli dihydrofolate reductase on stability and function: implication of hydrophobic interactions. Information

Substance Name: Tetrahydrofolate Dehydrogenase

Registry Number: EC 1.5.1.3

Grant and Affiliation Information for Effects of mutation at methionine-42 of Escherichia coli dihydrofolate reductase on stability and function: implication of hydrophobic interactions.

AFFILIATION: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima 739-8526.

Country: Japan

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MEDLINETA: J Biochem

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