Cysteine Synthase of an Extremely Thermophilic Bacterium, Thermus thermophilus HB8(Biochemistry & Molecular Biology)

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概要

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• O-Acetyl-L-serine sulfhydrylase (EC 4. 2. 99. 8) was first purified from an extremely thermophilic bacterium, Thermus thermophilus HB8, in order to ascertain that it is responsible for the cysteine synthesis in this organism cultured with either sulfate or methionine given as a sole sulfur source. Polyacrylamide gel electrophoreses both with and without SDS found high purity of the enzyme preparations finally obtained, through ammonium sulfate fractionation, ion exchange chromatography, gel filtration, and hydrophobic chromatography (or affinity chromatography). The enzyme activity formed only one elution curve in each of the four different chromatographies, strongly suggesting the presence of only one enzyme species in this organism. Molecular masses of 34,000 and 68,000 were estimated for dissociated subunit and the native enzyme, respectively, suggesting a homodimeric structure. The enzyme was stable at 70℃ at pH 7.8 for 60 min, and more than 90% of the activity was retained after incubation of its solution at 80℃ with 10 mM dithiothreitol. The enzyme was also quite stable at pH 8-12 (50℃, 30 min). It had an apparent K_m of 4.8 mM for O-acetyl-L-serine (with 1 mM sulfide) and a V_<max> of 435 μmol/min/mg of protein. The apparent K_m for sulfide was approximately 50 μM (with 20 mM acetylserine), suggesting that the enzyme can react with sulfide liberated very slowly from methionine. The absorption spectrum of the holo-enzyme and inhibition of the activity by carbonyl reagents suggested the presence of pyridoxal 5'-phosphate as a cofactor. The apo-enzyme showed an apparent K_m of 29 μM for the cofactor at pH 8. Monoiodoacetic acid (1 mM) almost completely inactivated the enzyme. The meaning of a very high enzyme content in the cell is discussed.

• 社団法人日本農芸化学会の論文
• 2002-03-23

著者

• MIYASHITA Yoko

  Department of Biology,Sapporo Medical College

• Miyashita Yoko

  Department Of Biology Sapporo Medical College

• Miyashita Yoko

  Department Of Biotechnology Faculty Of Agriculture Gifu University

• Mizuno Yasuko

  United Graduate School Of Agricultural Sciences Faculty Of Agriculture Gifu University

• YAMAGATA Shuzo

  United Graduate School of Agricultural Sciences Gifu University

• IWAMA Tomonori

  United Graduate School of Agricultural Sciences, Faculty of Agriculture, Gifu University

• AKAMATSU Tsuyoshi

  Department of Biotechnology, Faculty of Agriculture, Gifu University

• Yamagata Shuzo

  United Graduate School Of Agricultural Sciences Faculty Of Agriculture Gifu University:department Of

• Akamatsu Tsuyoshi

  Department Of Biotechnology Faculty Of Agriculture Gifu University

• Iwama Tomonori

  United Graduate School Of Agricultural Sciences Faculty Of Agriculture Gifu University:department Of

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