Role of residue 161 in the allosteric transitions of two bacterial phosphofructokinases.
Role of residue 161 in the allosteric transitions of two bacterial phosphofructokinases.
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残基 161 在两种细菌磷酸果糖激酶变构转变中的作用。
DOI：
10.1021/bi00021a018
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Chang,SH
中科院分区：
文献类型：
--
作者：
Auzat,I;Byrnes,WM;Garel,JR;Chang,SH
1995年3月20日收到的修订版摘要：来自嗜热脂肪芽孢杆菌的磷酸果糖激酶（PFK）的α-螺旋6和β-链F之间的环被认为在该酶的变构转变中是重要的[Schirmer，T.，& Evans，P. R.（1990）Nature 343，140 -145]，除了残基161，环内的氨基酸在B之间相似。嗜热脂肪菌PFK（BsPFK）和来自大肠杆菌的PFK（EcPFK）。在前一种酶中，残基161是谷氨酸，而在后一种酶中，它是谷氨酰胺。我们用定点突变研究了残基161对磷酸烯醇丙酮酸（PEP）（抑制剂）和GDP（激活剂）对这两种酶的变构调节的重要性。在BsPFK中，谷氨酸161已被改变为谷氨酰胺和丙氨酸，而在EcPFK中，谷氨酰胺161已被改变为谷氨酸、精氨酸和丙氨酸。突变酶的动力学参数与相应的野生型酶相似，表明残基161不直接参与底物结合和催化。EcPFK突变体Q161 A对PEP完全不敏感，但能被GDP正常激活。这表明残基161的氢键能力对于PEP抑制EcPFK是关键的，并且表明GDP激活和PEP抑制遵循EcPFK中不同的结构途径。BsPFK突变酶对PEP抑制不太敏感，对GDP激活更敏感，这表明抑制和激活是相反的，并且遵循与协调的变构机制一致的共同结构途径。
Revised Manuscript Received March 20, 1995s abstract: The loop between a-helix 6 and/3-strand F of the phosphofructokinase (PFK) from Bacillus stearothermophilus is proposedto be important in the allosteric transition of the enzyme [Schirmer, T., & Evans, P. R.(1990) Nature 343,140—145], Except for residue 161, the amino acids within the loop are similar between B. stearothermophilus PFK (BsPFK) and the PFK from Escherichia coli (EcPFK). In the former enzyme, residue 161 is a glutamate, while in the latter it is a glutamine. We haveused site-directed mutagenesisto investigate the importance of residue 161 for the allosteric regulation of the two enzymes by phosphoenolpyruvate (PEP), an inhibitor, and GDP, an activator. In BsPFK, glutamate 161 has been changed to a glutamine and an alanine, while in EcPFK, glutamine 161 has been changed to a glutamate, an arginine, and an alanine. The kinetic parameters of the mutant enzymes were similar to those of the respective wild types, indicating thatresidue 161 is not directly involved in substrate binding and catalysis. One of the EcPFK mutants, Q161A, though activatednormally by GDP, was completely insensitive toPEP. This indicates thatthe hydrogen-bonding abilityof residue 161 is critical for PEP inhibition of EcPFK and suggests that GDP activation and PEP inhibition follow different structural pathways in EcPFK. The BsPFK mutant enzymes were less sensitive to PEP inhibition and more sensitive to GDP activation, suggesting that inhibition and activation are opposed and follow a common structural pathway in agreement with a concerted allosteric mechanism.
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