Dement'eva E I, Zheleznova E E, Kutuzova G D, Lundovskikh I A, Ugarova N N
Faculty of Chemistry, M.V. Lomonosov Moscow State University.
Biokhimiia. 1996 Jan;61(1):152-9.
Physico-chemical properties of the recombinant L. mingrelica luciferase synthesized by E. coli cells have been studied. The catalytic and spectral properties of recombinant luciferase were similar to those of the native enzyme but the former was less stable in the presence of the additional Cys residue. The mutant forms of L. mingrelica firefly luciferase with point mutations Cys-82-->Ala, Cys-260-->Ala, Cys-393-->Ala and Thr-204-->Asp, have been constructed using the method of site-specific mutagenesis. Mutations Cys-82,260,393-->Ala changed slightly the Km values for ATP and luciferin but did not influence kcat. The Cys-393-->Ala mutant appeared to be more stable in comparison with the native enzyme. Mutation Thr-204-->Asp resulted in a 8-fold increase in the ATP binding constant and in a 2-fold increase in the kcat, thus indicating that Thr-204 may be located in the ATP-binding region of luciferase. Dithiothreitol, ethylene glycol, bovine serum albumin and trehalose had a stabilizing effect on the native, recombinant and mutant luciferases.
已对大肠杆菌细胞合成的重组明氏发光杆菌荧光素酶的物理化学性质进行了研究。重组荧光素酶的催化和光谱性质与天然酶相似，但前者在存在额外半胱氨酸残基的情况下稳定性较差。使用位点特异性诱变方法构建了具有点突变Cys-82→Ala、Cys-260→Ala、Cys-393→Ala和Thr-204→Asp的明氏发光杆菌萤火虫荧光素酶突变体形式。Cys-82、260、393→Ala突变略微改变了ATP和荧光素的Km值，但不影响kcat。与天然酶相比，Cys-393→Ala突变体似乎更稳定。Thr-204→Asp突变导致ATP结合常数增加8倍，kcat增加2倍，因此表明Thr-204可能位于荧光素酶的ATP结合区域。二硫苏糖醇、乙二醇、牛血清白蛋白和海藻糖对天然、重组和突变荧光素酶具有稳定作用。
