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首页> 外文期刊>Biochemistry >ISOTOPE PARTITIONING WITH ASCARIS SUUM PHOSPHOFRUCTOKINASE IS CONSISTENT WITH AN ORDERED KINETIC MECHANISM
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ISOTOPE PARTITIONING WITH ASCARIS SUUM PHOSPHOFRUCTOKINASE IS CONSISTENT WITH AN ORDERED KINETIC MECHANISM

机译：带有天花粉蛋白磷酸激酶的同位素分区与有序的动力学机制一致

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Isotope partitioning and initial velocity studies have been used to study the kinetic mechanism of Ascaris suum phosphofructokinase (PFK) at pH 8.0 for the native enzyme (nPFK), and at pH 6.8 for a form of enzyme desensitized (dPFK) to hysteresis in the reaction time course, to ATP allosteric inhibition, and to F6P homotropic cooperativity. Complete trapping (P*(max) approximate to 100%) of the E:MgATP* complex as fructose (1-P-32)-1,6-bisophosphate for both enzyme forms is consistent with the previously proposed steady-state ordered mechanism [Rao, G. S. J., Harris, B. G., & Cook, P. F. (1987) J. Biol. Chem. 262, 14074-14079] with MgATP binding before fructose 6-phosphate (F6P). K'(F6P) values for trapping of MgATP* of 0.54 +/- 0.09 mM for nPFK and 0.85 +/- 0.15 mM for dPFK were obtained. Saturating amounts of the heterotropic activator fructose 2,6-bisphosphate (F26P(2)) gives no change in the trapping parameters for nPFK with a P*(max) of 100% and a K'(F6P) of 0.40 +/- 0.06 mM. For dPFK, however, F26P(2) causes a decrease in both parameters, giving a P*(max) of 54% and a K'(F6P) of 0.26 +/- 0.07 mM. The partial trapping of E:MgATP* in the presence of F26P(2) for dPFK suggests that the activator changes the kinetic mechanism from an ordered to a random binding of substrates. Initial velocity studies confirm the change in mechanism. Uncompetitive inhibition by arabinose 5-phosphate (Ara5P), a dead-end inhibitory analog of F6P, versus MgATP for nPFK in the absence and presence of F26P(2) is consistent with an ordered mechanism with MgATP adding to enzyme prior to F6P. An uncompetitive pattern is also obtained with dPFK for Ara5P versus MgATP in the absence of F26P(2), but the pattern becomes noncompetitive in the presence of F26P(2), consistent with a change to a random mechanism. No trapping of the E:[C-14]F6P complex could be detected, indicating either that the E:[C-14]F6P complex does not form in a significant amount under the conditions used or that the off-rate for F6P from enzyme is much faster than the net rate constant for formation of the first product, FBP. The data are consistent with a predominantly ordered mechanism with MgATP binding prior to F6P. The minor pathway with MgATP dissociating from the E:F6P:MgATP ternary complex becomes apparent for the dPFK in the presence of F26P(2).
机译：同位素分配和初始速度研究已用于研究天然酶（nPFK）在pH 8.0时的A虫磷酸果糖激酶（PFK）的动力学机理，对于反应中的磁滞脱敏（dPFK）形式的酶对pH 6.8的动力学机理进行了研究。时间进程，对ATP的变构抑制作用，以及对F6P的同向性协同作用。两种酶形式的果糖（1-P-32）-1,6-二异磷酸盐完全捕获（P *（max）约100％）E：MgATP *复合物与先前提出的稳态有序机制一致[Rao，GSJ，Harris，BG，＆Cook，PF（1987）J. Biol。化学262，14074-14079]在果糖6-磷酸（F6P）之前具有MgATP结合。对于nPFK，捕获MgATP *的K'（F6P）值分别为0.54 +/- 0.09 mM和dPFK的0.85 +/- 0.15 mM。多相活化剂果糖2,6-二磷酸果糖（F26P（2））的饱和量对nPFK的俘获参数没有任何影响，P *（max）为100％，K'（F6P）为0.40 +/- 0.06毫米但是，对于dPFK，F26P（2）导致两个参数均减小，P *（max）为54％，K'（F6P）为0.26 +/- 0.07 mM。 E：MgATP *在dPFK的F26P（2）存在下的部分捕获表明，激活剂将动力学机制从有序结合变为随机结合。初始速度研究证实了机理的改变。在不存在和存在F26P（2）的情况下，阿拉伯糖5-磷酸（Ara5P）（F6P的末端抑制类似物）与MgATP对nPFK的非竞争性抑制作用与有序机制一致，即在F6P之前将MgATP添加到酶中。在不存在F26P（2）的情况下，对于Ara5P与MgATP，也可以通过dPFK获得非竞争性模式，但是在存在F26P（2）的情况下，该模式变为非竞争性，这与对随机机制的更改一致。没有检测到E：[C-14] F6P络合物的捕获，表明在所使用的条件下E：[C-14] F6P络合物没有大量形成，或者表明F6P的偏离速率酶比形成第一个产物FBP的净速率常数快得多。数据与在F6P之前具有MgATP结合的主要排序机制一致。在存在F26P（2）的情况下，对于dPFK，MgATP从E：F6P：MgATP三元复合物解离的次要途径变得很明显。

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