Reevaluation of the accepted allosteric mechanism of phosphofructokinase from Bacillus stearothermophilus
Reevaluation of the accepted allosteric mechanism of phosphofructokinase from Bacillus stearothermophilus
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DOI：
10.1073/pnas.050588097
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发表时间：
2000-04-11
影响因子：
11.1
通讯作者：
Reinhart, GD
中科院分区：
文献类型：
--
作者：
Kimmel, JL;Reinhart, GD
磷酸烯醇丙酮酸（PEP）与来自嗜热脂肪芽孢杆菌（BsPFK）的磷酸果糖激酶（EC 2.7.1.11）上的单一变构位点的结合降低了该酶结合底物果糖6-磷酸（Fru-6-P）的能力。与结合的Fru-6-P或磷酸乙醇酸盐（PEP的类似物）的晶体结构的比较已经显示Arg-162与带负电荷的Fru-6-P相互作用。在磷酸乙醇酸盐结合时，Arg-162实际上被Glu-161替代，这在酶和底物之间引入潜在的库仑排斥[Schirmer，T，& Evans，P. R.（1990）Nature（伦敦）343，140-145]，先前已经提出这种结构转变解释了BsPFK中的变构抑制，并且这种解释已经出现在教科书中以说明变构配体如何能够在一定距离处影响底物结合。已经使用定点诱变来产生BsPFK的三种突变体，其用丙氨酸残基取代Glu-161，Arg-162，或者两者都有。E161 A突变不影响PEP在25 ° C下对BsPFK的抑制，而R162 A突变使BsPFK对Fru-6-P的亲和力降低约30倍，R162 A使PEP抑制的有效性降低仅1/3。E161 A和R162 A组合产生与单独R162 A相当的行为。这些和其他数据表明，Glu-161和Arg-162的运动并不像Schirmer和Evans机制中最初设想的那样在PEP产生变构抑制中起核心作用。
The binding of phosphoenolpyruvate (PEP) to the single allosteric site on phosphofructokinase (EC 2.7.1.11) from Bacillus stearothermophilus (BsPFK) diminishes the ability of the enzyme to bind the substrate fructose 6-phosphate (Fru-6-P). Comparisons of crystal structures with either Fru-6-P or phosphoglycolate, an analog of PEP, bound have shown that Arg-162 interacts with the negatively charged Fru-6-P. Upon the binding of phosphoglycolate, Arg-162 is virtually replaced by Glu-161, which introduces a potential coulombic repulsion between enzyme and substrate [Schirmer, T, & Evans, P. R. (1990) Nature (London) 343, 140-145], It has previously been proposed that this structural transition explains the allosteric inhibition in BsPFK, and this explanation has appeared in textbooks to illustrate how an allosteric ligand can influence substrate binding at a distance, Site-directed mutagenesis has been employed to create three mutants of BsPFK that substitute an alanine residue for Glu-161, Arg-162, or both. The E161A mutation does not affect the inhibition of BsPFK by PEP at 25 degrees C, and while the R162A mutation decreases BsPFK's affinity for Fru-6-P by approximately 30-fold, R162A diminishes the effectiveness of PEP inhibition by only 1/3, Combining E161A and R162A produces behavior comparable to R162A alone. These and other data suggest that the movement of Glu-161 and Arg-162 does not play the central role in producing the allosteric inhibition by PEP as originally envisioned in the Schirmer and Evans mechanism.