Thermostable glycerol kinase from a hyperthermophilic archaeon: gene cloning and characterization of the recombinant enzyme.
Thermostable glycerol kinase from a hyperthermophilic archaeon: gene cloning and characterization of the recombinant enzyme.
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来自超嗜热古菌的热稳定性甘油激酶：重组酶的基因克隆和表征。
DOI：
10.1093/protein/11.12.1219
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
S. Kanaya
中科院分区：
文献类型：
--
作者：
Y. Koga;M. Morikawa;M. Haruki;H. Nakamura;T. Imanaka;S. Kanaya
Pk-glpK 基因编码来自超嗜热古菌 Kodakaraensis KOD1 的甘油激酶 (GK)，已在大肠杆菌中克隆并表达。从核苷酸序列推导的该酶(Pk-GK)的氨基酸序列与大肠杆菌GK的氨基酸序列有57％的同一性，与人GK的氨基酸序列有47％的同一性。 Pk-GK 的分子量为 55902（497 个氨基酸残基），从大肠杆菌中纯化并进行表征。尽管序列高度相似，Pk-GK 和大肠杆菌 GK 在结构和功能上彼此存在很大差异。与以四聚体形式存在的大肠杆菌 GK 不同，Pk-GK 以二聚体形式存在。 Pk-GK 的优选二价阳离子是 Co2+，而不是 Mg2+。 Pk-GK 活性的最佳 pH 值和温度分别为 8.0 和 80°C。 Pk-GK 可以利用除 ATP 之外的其他核苷三磷酸作为磷酰基供体。它对大肠杆菌 GK 的变构抑制剂 1,6-二磷酸果糖具有相当的抵抗力。动力学参数测定表明，该酶的ATP Km值为15.4 µM，甘油Km值为111 µM，kcat值为940 s(-1)。该酶对不可逆热失活具有相当的抵抗力，即使在 100 摄氏度加热 30 分钟后仍保留 50% 的酶活性。该酶三维结构模型的构建表明，广泛的离子对网络的形成是该酶具有高稳定性的原因。
The Pk-glpK gene, which encodes glycerol kinase (GK) from a hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1, was cloned and expressed in Escherichia coli. The amino acid sequence of this enzyme (Pk-GK) deduced from the nucleotide sequence showed 57% identity with that of E. coli GK and 47% identity with that of human GK. Pk-GK, which has a molecular weight of 55902 (497 amino acid residues), was purified from E. coli and characterized. Despite the high sequence similarity, Pk-GK and E. coli GK are greatly divergent in structure and function from each other. Unlike E. coli GK, which exists as a tetramer, Pk-GK exists as a dimer. The preferred divalent cation for Pk-GK is Co2+, instead of Mg2+. The optimum pH and temperature for Pk-GK activity are 8.0 and 80 degrees C, respectively. Pk-GK can utilize other nucleoside triphosphates than ATP as a phosphoryl donor. It is fairly resistant to an allosteric inhibitor of E. coli GK, fructose-1,6-bisphosphate. Determination of the kinetic parameters indicates that the Km value of the enzyme is 15.4 microM for ATP and 111 microM for glycerol and its kcat value is 940 s(-1). The enzyme was shown to be fairly resistant to irreversible heat inactivation and still retained 50% of its enzymatic activity even after heating at 100 degrees C for 30 min. Construction of a model for the three-dimensional structure of the enzyme suggests that the formation of extensive ion-pair networks is responsible for the high stability of this enzyme.