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Biochemical characterization of a thermostable cysteine synthase from Geobacillus stearothermophilus V

机译:嗜热嗜热脂肪地芽孢杆菌V的热稳定半胱氨酸合酶的生化特性

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The cysK gene encoding a cysteine synthase of Geobacillus stearothermophilus V was overexpressed in E coli and the recombinant protein was purified and characterized. The enzyme is a thermostable homodimer (32 kDa/monomer) belonging to the beta family of pyridoxal phosphate (PLP)-dependent enzymes. UV-visible spectra showed absorption bands at 279 and 410 nm. The band at 279 nm is due to tyrosine residues as the enzyme lacks tryptophan. The 410 nm band represents absorption of the coenzyme bound as a Schiff base to a lysine residue of the protein. Fluorescence characteristics of CysK's Schiff base were influenced by temperature changes suggesting different local structures at the cofactor binding site. The emission of the Schiff base allowed the determination of binding constants for products at both 20 degreesC and 50 degreesC. At 50 degreesC and in the absence of sulphide the enzyme catalyzes the decomposition of O-acetyl-L-serine to pyruvate and ammonia. At 20 degreesC, however, a stable a-aminoacrylate intermediate is formed. (C) 2004 Elsevier SAS. All rights reserved.
机译:在大肠杆菌中过表达编码嗜热脂肪地芽孢杆菌V的半胱氨酸合酶的cysK基因,并纯化和鉴定了重组蛋白。该酶是热稳定的同型二聚体(32 kDa /单体),属于磷酸吡ido醛(PLP)依赖性酶的β家族。紫外可见光谱显示在279和410 nm处的吸收带。 279 nm处的条带归因于酪氨酸残基,因为该酶缺乏色氨酸。 410 nm谱带代表辅酶作为席夫碱与蛋白质的赖氨酸残基结合的吸收。 CysK的席夫碱的荧光特性受温度变化的影响,表明辅因子结合位点的局部结构不同。席夫碱的发射使得可以确定在20℃和50℃下产物的结合常数。在50摄氏度且不存在硫化物的条件下,该酶催化O-乙酰基-L-丝氨酸分解为丙酮酸和氨。然而,在20℃下,形成稳定的α-氨基丙烯酸酯中间体。 (C)2004 Elsevier SAS。版权所有。

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