Takasaki Y, Kitajima Y, Takahashi I, Sakakibara M, Mukai T, Hori K
Department of Biochemistry, Saga Medical School, Japan.
Prog Clin Biol Res. 1990;344:935-53.
Enzymatic studies on aldolase isozymes have been carried out by techniques of protein engineering. Site-directed mutagenesis helps us to verify the roles of amino acid residues in catalytic reactions. Chimeric fusion proteins give us information about the regions which specify the characteristics of the isozymes. The results are: (1) In aldolase A, COOH terminal Tyr and Lys-107 residues play important roles in catalysis, especially in binding of FDP. (2) Aspartic acid at the 128th residue in aldolase A is essential to thermostability; no other residue such as glutamic acid can substitute for it. (3) Studies on chimeric fusion proteins indicate that the C-terminal region (including C-terminus Tyr) or aldolase A is responsible for its substrate specificity, which is not seen in aldolase B. (4) A region near NH2 terminus in aldolase B determines its specific structure. (5) The region including His-107, Asp-128, and Tyr-137 (B-A junction of BA137) is located in a turn which is exposed outward (a model architecture by Sygusch et al [1987]). In BA137, this region would be constrained, and play a significant role in catalysis, thermostability, etc. (6) Tertiary structure of aldolase B seems to be dissimilar to that of aldolase A.
已通过蛋白质工程技术对醛缩酶同工酶进行了酶学研究。定点诱变有助于我们验证氨基酸残基在催化反应中的作用。嵌合融合蛋白为我们提供了有关决定同工酶特性区域的信息。结果如下：（1）在醛缩酶A中，COOH末端的酪氨酸和第107位赖氨酸残基在催化中起重要作用，尤其是在与1,6-二磷酸果糖（FDP）的结合中。（2）醛缩酶A中第128位的天冬氨酸对热稳定性至关重要；没有其他残基如谷氨酸可以替代它。（3）对嵌合融合蛋白的研究表明，C末端区域（包括C末端酪氨酸）或醛缩酶A负责其底物特异性，这在醛缩酶B中未见。（4）醛缩酶B中NH2末端附近的一个区域决定了其特定结构。（5）包括组氨酸-107、天冬氨酸-128和酪氨酸-137（BA137的B-A连接点）的区域位于一个向外暴露的转角处（Sygusch等人[1987]的模型结构）。在BA137中，该区域将受到限制，并在催化、热稳定性等方面发挥重要作用。（6）醛缩酶B的三级结构似乎与醛缩酶A不同。
