Identification of substrate contact residues important for the allosteric regulation of phosphofructokinase from Escherichia coli
Identification of substrate contact residues important for the allosteric regulation of phosphofructokinase from Escherichia coli
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DOI：
10.1021/bi034273t
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发表时间：
2003-06-03
期刊：
影响因子：
2.9
通讯作者：
Reinhart, GD
中科院分区：
文献类型：
--
作者：
Fenton, AW;Paricharttanakul, NM;Reinhart, GD
检查与结合底物果糖 6-磷酸 (Fru-6-P) 相互作用的大肠杆菌磷酸果糖激酶 (EcPFK) 侧链在变构调节中的潜在作用。除催化碱基 D127 外，在每个接触残基处都会产生严重降低 Fru-6-P 亲和力和/或 k(cat)/K-m 的突变。尽管 Fru-6-P 对 R162E、M169A、E222A/H223A 和 R243E 的亲和力大大降低，但突变蛋白保留了被 MgADP 激活并被磷酸烯醇丙酮酸 (PEP) 抑制的能力。 R252E 没有表现出对 MgADP 或 PEP 的变构反应。 H249E 突变保留了 MgADP 激活，但对 PEP 没有反应。 R72E、T125A 和 R171E 维持 PEP 的变构抑制。 R72E 和 T125A 均表现出 MgADP 依赖性 k(cat) 降低，但没有 MgADP 依赖性 K 型效应。 R171E 维持 MgADP 依赖性 K 型激活，但也显示出 MgADP 依赖性 k(cat) 降低。改变转移磷酸基团附近 MgADP 激活的突变定位表明 Fru-6-P 的 I-甲氧基区域在 MgADP 变构调节中的重要性。 6'-磷酸附近的区域对于 PEP 抑制可能同样重要。 R252 独特地位于结合的 Fru-1,6-BP 的 1'- 和 6'-磷酸之间，该位置的突变可能会改变两个变构反应区域。 Fru-6-P 接触残基中特定区域的差异功能支持不同的变构激活和抑制机制。此外，降低 Fru-6-P 亲和力的突变与废除变构通讯的突变之间缺乏相关性，支持了亲和力和变构偶联的独立性。
The side chains of Escherichia coli phosphofructokinase (EcPFK) that interact with bound substrate, fructose 6-phosphate (Fru-6-P), are examined for their potential roles in allosteric regulation. Mutations that severely decrease Fru-6-P affinity and/or k(cat)/K-m, were created at each contact residue, with the exception of the catalytic base, D127. Even though Fru-6-P affinity was greatly decreased for R162E, M169A, E222A/H223A, and R243E, the mutated proteins retained the ability to be activated by MgADP and inhibited by phosphoenolpyruvate, (PEP). R252E did not show an allosteric response to either MgADP or PEP. The H249E mutation retained MgADP activation but did not respond to PEP. R72E, T125A, and R171E maintained allosteric inhibition by PEP. Both R72E and T125A displayed a MgADP-dependent decrease in k(cat) but no MgADP-dependent K-type effects. R171E maintained MgADP-dependent K-type activation but also displayed a MgADP-dependent decrease in k(cat). Localization of mutations that alter MgADP activation near the transferred phosphate group indicates the importance of the I-methoxy region of Fru-6-P in allosteric regulation by MgADP. A region near the 6'-phosphate may be similarly important for PEP inhibition. R252 is uniquely positioned between the 1'- and 6'-phosphates of bound Fru-1,6-BP, and the mutation at this position may alter both allosterically responsive regions. The differential functions of specific regions in the Fru-6-P contact residues support different mechanisms for allosteric activation and inhibition. In addition, the lack of correlation between mutations that decrease Fru-6-P affinity and those that abolish allosteric communications supports the independence of affinity and allosteric coupling.