Kusakabe T, Motoki K, Hori K
Department of Biochemistry, Saga Medical School.
J Biochem. 1994 Jun;115(6):1172-7. doi: 10.1093/oxfordjournals.jbchem.a124475.
To study the structure/function relationship and enzymatic properties of human aldolase C, we have constructed an Escherichia coli expression plasmid, pHAC11, for the isozyme. E. coli cells carrying this plasmid produced enzymatically active human aldolase C. The kcat and Km values for fructose-1,6-bisphosphate (Fru-1,6-P2) and fructose-1-phosphate (Fru-1-P) of the recombinant enzyme were found to be similar to those of authentic aldolase C from human brain. The Fru-1,6-P2/Fru-1-P activity ratio of the recombinant enzyme is approximately 13.5, which is comparable to that of the recombinant rat aldolase C, but is slightly higher than those of rat brain and hepatoma aldolases C. The substitution of Ser for the carboxyl-terminal Tyr (Tyr-363) of the recombinant enzyme caused a marked decrease in that of Fru-1,6-P2, with little change in that of Fru-1-P. The activity ratio changed from 13.5 for the normal enzyme to 3.8 for the engineered enzyme. Human aldolase C was found to form tetrameric hybrids with aldolase B in vivo when these enzymes were coexpressed in E. coli cells.
为了研究人醛缩酶C的结构/功能关系和酶学性质，我们构建了一种用于该同工酶的大肠杆菌表达质粒pHAC11。携带此质粒的大肠杆菌细胞产生了具有酶活性的人醛缩酶C。发现重组酶对1,6-二磷酸果糖（Fru-1,6-P2）和1-磷酸果糖（Fru-1-P）的kcat和Km值与来自人脑的天然醛缩酶C相似。重组酶的Fru-1,6-P2/Fru-1-P活性比约为13.5，与重组大鼠醛缩酶C相当，但略高于大鼠脑和肝癌醛缩酶C。将重组酶羧基末端的酪氨酸（Tyr-363）替换为丝氨酸导致Fru-1,6-P2的活性显著降低，而Fru-1-P的活性变化不大。活性比从正常酶的1,6-二磷酸果糖变化为工程酶的3.8。当这些酶在大肠杆菌细胞中共表达时，发现人醛缩酶C在体内与醛缩酶B形成四聚体杂种。
