Cysteine Synthase of an Extremely Thermophilic Bacterium, Thermus thermophilus HB8
Cysteine Synthase of an Extremely Thermophilic Bacterium, Thermus thermophilus HB8
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极嗜热细菌（嗜热栖热菌 HB8）的半胱氨酸合成酶
DOI：
10.1271/bbb.66.549
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
Tsuyoshi Akamatsu
中科院分区：
文献类型：
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作者：
Y. Mizuno;Y. Miyashita;S. Yamagata;T. Iwama;Tsuyoshi Akamatsu
O-Acetyl-L-serine sulfhydrylase （EC 4.2.99.8）首先从一种极端嗜热细菌Thermus thermophilus HB8中纯化出来，以确定它在以硫酸盐或蛋氨酸作为唯一硫源培养的细菌中负责半胱氨酸的合成。经过硫酸铵分馏法、离子交换层析法、凝胶过滤法和疏水层析法（或亲和层析法），无论加SDS还是不加SDS的聚丙烯酰胺凝胶电泳均可获得纯度较高的酶制剂。在四种不同的色谱中，酶活性仅形成一条洗脱曲线，强烈表明该生物中仅存在一种酶。估计解离亚基和天然酶的分子质量分别为34,000和68,000，表明其具有同二聚体结构。该酶在70℃pH 7.8条件下稳定60 min，其溶液与10 mM二硫苏糖醇在80℃条件下孵育可保持90%以上的活性。该酶在pH 8-12（50°C, 30 min）下也很稳定。对o -乙酰- l-丝氨酸（含1 mM硫化物）的表观K m为4.8 mM，最大V值为435 μmol/min/mg蛋白质。硫化物的表观K m约为50 μM（含20 mM乙酰丝氨酸），表明该酶可以与蛋氨酸中缓慢释放的硫化物反应。全酶的吸收光谱和羰基试剂对活性的抑制表明5′-磷酸吡哆醛作为辅助因子存在。在pH为8时，载脂蛋白酶的表观K m为29 μM。单碘乙酸（1mm）几乎完全使酶失活。讨论了细胞中酶含量非常高的意义。
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°C at pH 7.8 for 60 min, and more than 90% of the activity was retained after incubation of its solution at 80°C with 10 mM dithiothreitol. The enzyme was also quite stable at pH 8–12 (50°C, 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.
半胱氨酸 42 对于维持 O-乙酰丝氨酸硫化氢解酶的完整活性位点非常重要，从而导致 α-氨基丙烯酸酯中间体的稳定。
DOI：
10.1021/bi980647k
发表时间：
1998
期刊：
Biochemistry.
影响因子：
--
作者：
Tai,CH;Yoon,MY;Kim,SK;Rege,VD;Nalabolu,SR;Kredich,NM;Schnackerz,KD;Cook,PF
通讯作者：
Cook,PF