大肠杆菌Ⅱ型丙酮酸激酶的纯化分析及其作为偶联酶的应用  

Purification and Characterization of Pyruvate Kinase Ⅱ from Escherichia coli and Its Application as a Coupling Enzyme

作　　者：李鸿艳[1] 周思甜[1] 马建辉[1] 王艳兴[1,2] 孙梅好[1] 

机构地区：[1]浙江师范大学化学与生命科学学院,金华321004 [2]中国科学院上海生命科学研究院生物化学与细胞生物学研究所,上海200031

出　　处：《中国生物工程杂志》2013年第5期68-74,共7页China Biotechnology

摘　　要：目的:分析大肠杆菌Ⅱ型丙酮酸激酶(pyruvate kinase Ⅱ,PKⅡ)的酶学特性及其作为偶联酶的可行性。方法:PCR法从大肠杆菌基因组克隆pkⅡ,构建入原核表达载体pSKB4,经IPTG诱导表达、纯化获得PKⅡ。利用乳酸脱氢酶偶联法分析其催化ADP、GDP、UDP、CDP磷酸化的能力以及不同pH、温度对其活性的影响,并利用PKⅡ作为偶联酶对球形红细菌硫酸盐活化复合体(sulfate activating complex,SAC)及其突变体进行了活性分析。结果:原核表达、纯化、分析大肠杆菌PKⅡ,发现其对ADP、GDP、UDP的催化效率与兔肌肉PK类似,且由大到小分别为:A>G>C>U;而对CDP的催化效率为兔肌肉PK的6倍。PKⅡ的最适pH为7.0,在20～50℃范围内活性稳定,且18个月的-80℃低温及24 hr的室温存放基本不影响其活性。PKII的高催化效率以及稳定性表明其可作为偶联酶进行NDP的测定。利用PKⅡ作为偶联酶对球形红细菌SAC及其突变体的活性测定表明S410和K409参与了5'-腺苷磷酰硫酸的磷酸化。Pyruvate kinase （ PK）, catalyzes the transfer of the phosphoryl group from phosphoenolpyruvate to ADP during glycolysis, which step is essentially irreversible under intracellular conditions and is allosterically controlled. In Escherichia coli, there are two kinds of PKs, namely PKI （PKF） and PKII （PKA） which were activated by fructose 1,6-bisphosphate and adenosine 5＇-monophosphate, respectively. PKI had been extensively studied, while PKII had little been investigated largely due to its instability reported by Waywood et al. in 1975. To further characterize PKII, it was prokaryotically overexpressed and purified. Following the determination of kinetic constants for NDP, their catalytic efficiencies （CEs） were calculated and compared. PKII and PK from rabbit muscle （PKr） had similar CEs respect to ADP, GDP and UDP, and PKII had 6.5 times higher CE with CDP than that for PKr. Distinct from published results, little activity decrease was found during 18 months -80℃ storage or 24 h room temperature treatment. High catalytic efficiency and stability enabled PKⅡ as a potentially good coupling enzyme. Together with lactate dehydrogenase, PKⅡwas successfully used as coupling enzyme to determine the kinetic constants of sulfate activating complex （SAC） from Rhodobacter sphaeroides and its mutants, and K409 and S410 were found to be essential for its activity of APSK domain.

关 键 词：丙酮酸激酶 硫酸盐活化复合体 大肠杆菌 球形红细菌 

分 类 号：Q555.7[生物学—生物化学]