Role of glycine 212 in the allosteric behavior of phosphofructokinase from Bacillus stearothermophilus.
Role of glycine 212 in the allosteric behavior of phosphofructokinase from Bacillus stearothermophilus.
复制标题
甘氨酸 212 在嗜热脂肪芽孢杆菌磷酸果糖激酶变构行为中的作用。
DOI：
10.1021/bi00008a021
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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：
Zhu,X;Byrnes,M;Nelson,JW;Chang,SH
摘要：晶体学研究表明，与嗜热脂肪芽孢杆菌磷酸果糖激酶（BsPFK）的效应位点接壤的α-螺旋8和β-链H之间的环（8H环）参与酶的变构机制[Schirmer，T.，和Evans，P. R.（1990）Nature 343，140-145]。在这个环的一端的残基，甘氨酸212，被认为是环铰链的枢轴。使用定点诱变，甘氨酸212被替换为缬氨酸（G212 V）。稳态动力学分析和配体结合研究的改变和本地PFKs表明，G212 V取代导致在效应位点的可辨别的变化。突变的PFK需要比天然酶高3倍的变构抑制剂磷酸烯醇丙酮酸浓度才能引起相同水平的抑制。改变的PFK具有比野生型酶高2倍的别构激活剂GDP的解离常数。更重要的是，而nativePFK完全激活1 mM GDP从PEP抑制的T-状态，改变的酶只有轻微激活。另一方面，G212 V突变导致BsPFK的催化位点没有变化。催化速率常数&cat保持不变。改变后的PFK对ATP和果糖-6-磷酸（Fru-6-P）的Km值与野生型酶相同。此外，从相同的PEP抑制的T状态开始，两种酶对增加的Fru-6-P浓度给出相同的S形响应，这表明Fru-6-P可以将两者激活为R-状态。变构酶的活性可以通过在活性位点中结合协同底物进行同向性调节，并且通过在效应位点中结合变构效应物进行异向性调节[Perutz（1990）]。这种调节的一个例子是糖酵解酶磷酸果糖激酶（PFK，1* 56 EC 2.7）。1.11），催化ATP依赖的磷酸化，
Revised Manuscript Received November 30, 1994® abstract: Crystallographic studies indicate that the loop between a-helix 8 and/3-strand H (the 8H loop) which borders the effector site of Bacillus stearothermophilus phosphofructokinase (BsPFK) is involved in the allosteric mechanism of the enzyme [Schirmer, T., and Evans, P. R.(1990) Nature 343, 140—145]. The residue at one end of this loop, glycine 212, has beenproposed to be a pivot about which the loop hinges. Using site-directed mutagenesis, glycine 212 was replaced with valine (G212V). Steady-state kinetic analysis and ligand binding studies on the altered and native PFKs showed that the G212V substitution resulted in discernible changes at the effector site. The mutated PFK required a 3-fold higher concentration of the allosteric inhibitor phosphoenolpyruvate than did the native enzymeto cause the same level of inhibition. The altered PFK had a 2-fold higher dissociation constant for the allosteric activator GDP than the wild-type enzyme. More importantly, whereas the nativePFK was fully activated by 1 mM GDP from its PEP-inhibited T-state, the altered enzyme was only marginally activated. On the other hand, the G212V mutation resulted in no changes at the catalytic site of BsPFK. The catalytic rate constant &cat remained unchanged. The altered PFK had the same Km values for ATP and fructose-6-phosphate (Fru-6-P) as did the wild-type enzyme. Furthermore, starting from the same PEP-inhibited T-state, both enzymes gave identical sigmoidal responses to increasing Fru-6-P concentration, indicating that Fru-6-P can activate both to the R-state.The activity of an allosteric enzyme can be regulated homotropically through the binding of a cooperative substrate in the active site and heterotropic ally by the binding of allosteric effectors in the effector site [reviewed by Perutz (1990)]. An example of such regulation is seen in the glycolytic enzyme phosphofructokinase (PFK, 1*** 56EC 2.7. 1.11), which catalyzesthe ATP-dependent phosphorylation of