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首页> 外文期刊>Biochemistry >MgATP-dependent activation by phosphoenolpyruvate of the E187A mutant of escherichia coli phospohfructokinase
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MgATP-dependent activation by phosphoenolpyruvate of the E187A mutant of escherichia coli phospohfructokinase

机译：磷酸烯醇丙酮酸对大肠杆菌磷酸果糖激酶E187A突变体的MgATP依赖性激活

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Using enzymatic assays and steady-state fluorescence emission, we performed a linkage analysis of the three-ligand interaction of fructose 6-phosphate (Fru-6-P), phosphoenolpyruvate (PEP), and MgATP on El 87A mutant Escherichia ccli phosphofructokinase (PFK). PEP allosterically inhibits Fru-6-P binding to F. ccli PFK. The magnitude of antagonism is 90-fold in the absence and 60-fold in the presence of a saturating concentration of MgATP [Johnson, J. J., and Reinhart, C. D. (1997) Biochemistiy 36, 12814— 12822]. Substituting an alanine for the glutamate at position 187, located in the allosteric site (i.e., mutant E187A), activates Fru-6-P binding and inhibits the maximal rate of enzyme turnover [Lau, F. T.-K., and Fersht, A. R. (1987) Nature 326, 811—812]. The allosteric action of PEP appears to depend on the presence of the cosubstrate MgATP. In the presence of a saturating concentration of MgATP, PEP enhances the binding of Fru-6-P to the enzyme by a modest 2-fold. Decreasing the concentration of MgATP mitigates the extent of activation. At MgATP concentrations approaching 25 ~uM, PEP becomes insensitive to the binding of Fru-6-P. At MgATP concentrations <25 yM, PEP “crosses over” and becomes antagonistic toward substrate binding. The present study examines the role of Glu 187 at the allosteric site in the binding of Fru-6-P and offers a more complex explanation of the mechanism than that described by traditional allosteric mechanistic models.
机译：使用酶分析和稳态荧光发射，我们对果糖6-磷酸（Fru-6-P），磷酸烯醇丙酮酸（PEP）和MgATP在El 87A突变型大肠杆菌ccli磷酸果糖激酶（PFK）上的三配体相互作用进行了连锁分析。 ）。 PEP变构抑制Fru-6-P与F. ccli PFK的结合。在不存在饱和浓度的MgATP的情况下，拮抗作用的强度为90倍，在存在条件下为60倍[Johnson，J. J.和Reinhart，C. D.（1997）Biochemistiy 36，12814-12822]。用丙氨酸代替变构位点（即突变体E187A）上第187位的谷氨酸，可激活Fru-6-P结合并抑制最大酶转化率[Lau，FT-K。和Fersht，AR（ 1987）Nature 326，811-812]。 PEP的变构作用似乎取决于共底物MgATP的存在。在饱和浓度的MgATP存在下，PEP将Fru-6-P与酶的结合程度提高2倍。降低MgATP的浓度会降低活化程度。当MgATP浓度接近25μM时，PEP对Fru-6-P的结合变得不敏感。在MgATP浓度<25 yM时，PEP“越过”并变成对底物结合的拮抗作用。本研究检查了变构位点的Glu 187在Fru-6-P结合中的作用，并提供了比传统变构机理模型更为复杂的机理解释。

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