Site-directed mutagenesis in Bacillus stearothermophilus fructose-6-phosphate 1-kinase. Mutation at the substrate-binding site affects allosteric behavior.
Site-directed mutagenesis in Bacillus stearothermophilus fructose-6-phosphate 1-kinase. Mutation at the substrate-binding site affects allosteric behavior.
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发表时间：
1989-01
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通讯作者：
B. Valdez;B. A. French;E. S. Younathan;S. Chang
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作者：
B. Valdez;B. A. French;E. S. Younathan;S. Chang
嗜热脂肪芽孢杆菌果糖 6-磷酸 1-激酶 (PFK) 的 Arg252 被认为参与底物 Fru-6-P 的结合。我们在此证明，该残基突变为丙氨酸可将酶转化为与其变构紧密形式具有相似特征的形式。该突变酶对其抑制剂磷酸烯醇丙酮酸表现出高亲和力（与野生型相比有 68 倍的差异），并且 Fru-6-P 亲和力显着降低（Km 增加了 1500 倍）。与野生型相比，它对高 ATP 浓度的抑制更敏感，并且 ADP、GDP 或更高浓度的 Fru-6-P 可以缓解这种抑制。相比之下，Arg252 突变为赖氨酸，酶对 P-烯醇丙酮酸的亲和力仅增加 2 倍，对 Fru-6-P 的 Km 仅增加 50 倍。在 P-烯醇丙酮酸存在下观察到 Fru-6-P 的 S 形动力学，野生型嗜热脂肪芽孢杆菌 PFK 以及 Arg252 赖氨酸和丙氨酸突变的 Hill 数分别为 2.2、2.4 和 1.7。与大肠杆菌中的果糖 6-磷酸 1-激酶不同，在缺乏 P-烯醇丙酮酸的情况下，嗜热脂肪芽孢杆菌 PFK 的 Fru-6-P 浓度表现出双曲线特征。嗜热脂肪芽孢杆菌 PFK 对高 ATP 浓度的抑制敏感，并且受 GDP 或 ADP 竞争性抑制。我们的数据表明，嗜热脂肪芽孢杆菌 PFK 的 Arg252 在 Fru-6-P 结合和亚基之间的变构相互作用中发挥着重要作用。然而，该残留物似乎并不直接参与催化过程。
Arg252 of fructose-6-phosphate 1-kinase (PFK) from Bacillus stearothermophilus has been proposed to be involved in the binding of the substrate Fru-6-P. We demonstrate here that mutation of this residue to alanine converts the enzyme to a form with characteristics similar to those of its allosterically tight form. The mutant enzyme exhibits a high affinity for its inhibitor phosphoenolpyruvate (a 68-fold difference compared to wild type) and a dramatically decreased Fru-6-P affinity (1500-fold increase in Km). It is more sensitive to inhibition by high ATP concentrations than the wild type, and this inhibition is relieved by ADP, GDP, or higher Fru-6-P concentrations. In contrast, mutation of Arg252 to lysine increases the affinity of the enzyme for P-enolpyruvate by only 2-fold and increases its Km for Fru-6-P by only 50-fold. Sigmoidal kinetics with respect to Fru-6-P in the presence of P-enolpyruvate were observed with Hill numbers of 2.2, 2.4, and 1.7 for wild-type B. stearothermophilus PFK and the Arg252 to lysine and to alanine mutations, respectively. Unlike fructose-6-phosphate 1-kinase from Escherichia coli, in the absence of P-enolpyruvate, B. stearothermophilus PFK exhibits a hyperbolic profile with respect to Fru-6-P concentration. B. stearothermophilus PFK is sensitive to inhibition by high ATP concentrations and competitively inhibited by GDP or ADP. Our data indicate that Arg252 of B. stearothermophilus PFK plays a major role in both Fru-6-P binding and allosteric interaction between the subunits. However, this residue does not seem to participate directly in the catalytic process.