KINETIC CHARACTERISTICS OF PHOSPHOFRUCTOKINASE FROM BACILLUS-STEAROTHERMOPHILUS - MGATP NONALLOSTERICALLY INHIBITS THE ENZYME
KINETIC CHARACTERISTICS OF PHOSPHOFRUCTOKINASE FROM BACILLUS-STEAROTHERMOPHILUS - MGATP NONALLOSTERICALLY INHIBITS THE ENZYME
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DOI：
10.1021/bi00177a036
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发表时间：
1994-03-22
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：
BYRNES, M;ZHU, XM;CHANG, SH
用稳态测量方法研究了嗜热芽孢杆菌磷酸果糖激酶的动力学机制。反向反应的初始速度、产物抑制和混合交替底物研究的双倒数模式表明，该机制包括底物的快速平衡随机结合和由酶、镁ADP和果糖6-磷酸组成的流产络合物(E-MgADP-Fu-6P)的形成。正向反应的初始速度模式显示出显著的非线性，类似于竞争底物(MgATP)对服从随机机制的酶的抑制。突变的BsPFK酶(GV212)被用来证明这种抑制不是由于效应部位的镁结合所致。在考虑了镁ATP对底物抑制的影响后，对正向反应的产物和死端抑制研究符合随机机理。在低浓度下的初始速度测量表明，镁ATP的结合不是一个快速平衡的过程，即催化速度快于底物结合速度。结果表明，正向反应的动力学机制是顺序随机的，结合速率慢于催化速率。提出了一个结合了这些结果的模型，并提出了底物结合通过两条可选的途径进行，其中一条在动力学上是不利的。观察到的镁ATP底物抑制来自于通过不利途径的反应通量，以及在某种程度上，镁ATP在Fru-6P位点的结合失败。
The kinetic mechanism of phosphofructokinase from Bacillus sterothermophilus has been investigated using steady-state measurements. The double-reciprocal patterns observed for initial velocity, product inhibition, and mixed alternate substrate studies of the reverse reaction establish that the mechanism involves rapid-equilibrium random binding of substrates and the formation of an abortive complex composed of enzyme, MgADP, and fructose 6-phosphate (E-MgADP-Fru-6P). Initial velocity patterns for the forward reaction show significant nonlinearity and resemble those seen for competitive substrate (MgATP) inhibition of an enzyme that obeys a random mechanism. A mutant BsPFK enzyme (GV212) was used to show that the inhibition is not due to MgATP binding in the effector site. Product and dead-end inhibition studies of the forward reaction are consistent with a random mechanism, after taking into account the effects of substrate inhibition by MgATP. Initial velocity measurements at low MgATP concentration show that the binding of MgATP is not a rapid-equilibrium process; i.e., the rate of catalysis is faster than the rate of substrate binding. It is concluded that the kinetic mechanism of the forward reaction is sequential random, with the rate of MgATP binding slower than the catalytic rate. A model is presented that incorporates these results and proposes that substrate binding proceeds through two alternative pathways, one of which is kinetically disfavored. The observed MgATP substrate inhibition arises from both reaction flux through the disfavored pathway and, to some extent, abortive binding of MgATP in the Fru-6P site.