Soh Y, Song B J, Jeng J, Kallarakal A T
Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, 12501 Washington Avenue, Rockville, MD 20852, USA.
Biochem J. 1998 Jul 15;333 ( Pt 2)(Pt 2):367-72. doi: 10.1042/bj3330367.
It has been shown that one arginine per monomer at an unknown position is essential for enzyme activity of the homodimeric transketolase (TK) [Kremer, Egan and Sable (1980) J. Biol. Chem. 255, 2405-2410]. To identify the critical arginine, four highly conserved arginine residues of rat TK (Arg102, Arg350, Arg433 and Arg506) were replaced with alanine by site-directed mutagenesis. Wild-type and mutant TK proteins were produced in Escherichia coli and characterized. The Arg102-->Ala mutant exhibited similar catalytic activity to the wild-type enzyme, whereas Arg350-->Ala, Arg506-->Ala and Arg433-->Ala mutants exhibited 36.7, 37.0 and 6.1% of the wild-type activity respectively. Three recombinant proteins (wild-type, Arg350-->Ala and Arg433-->Ala) were purified to apparent homogeneity using Ni2+-affinity chromatography and further characterized. All these proteins were able to form homodimers (148 kDa), as shown by immunoblot analysis subsequent to non-denaturing gel electrophoresis. The Arg433-->Ala mutant protein was less stable than the wild-type and Arg350-->Ala proteins at 55 degrees C. Kinetic analyses revealed that both Vmax and Km values were markedly affected in the Arg433-->Ala mutant. The Km values for two substrates xylulose 5-phosphate and ribose 5-phosphate were 11.5- and 24.3-fold higher respectively. The kcat/Km values of the Arg433-->Ala mutant for the two substrates were less than 1% of those of the wild-type protein. Molecular modelling of the rat TK revealed that Arg433 of one monomer has three potential hydrogen-bond interactions with the catalytically important highly conserved loop of the other monomer. Thus, our biochemical analyses and modelling data suggest the critical role of the previously uncharacterized Arg433 in TK activity.
已表明，同二聚体转酮醇酶（TK）的每个单体中一个位置未知的精氨酸对于酶活性至关重要[克雷默、伊根和萨布尔（1980年）《生物化学杂志》255卷，2405 - 2410页]。为了鉴定关键的精氨酸，通过定点诱变将大鼠TK的四个高度保守的精氨酸残基（Arg102、Arg350、Arg433和Arg506）替换为丙氨酸。野生型和突变型TK蛋白在大肠杆菌中产生并进行表征。Arg102→Ala突变体表现出与野生型酶相似的催化活性，而Arg350→Ala、Arg506→Ala和Arg433→Ala突变体分别表现出野生型活性的36.7%、37.0%和6.1%。使用镍离子亲和层析将三种重组蛋白（野生型、Arg350→Ala和Arg433→Ala）纯化至表观均一，并进一步表征。如非变性凝胶电泳后的免疫印迹分析所示，所有这些蛋白都能够形成同二聚体（148 kDa）。在55℃时，Arg433→Ala突变体蛋白比野生型和Arg350→Ala蛋白更不稳定。动力学分析表明Arg433→Ala突变体的Vmax和Km值均受到显著影响。两种底物5 - 磷酸木酮糖和5 - 磷酸核糖的Km值分别高出11.5倍和24.3倍。Arg
