Citrate inhibition-resistant form of 6-phosphofructo-1-kinase from Aspergillus niger
Citrate inhibition-resistant form of 6-phosphofructo-1-kinase from Aspergillus niger
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DOI：
10.1128/aem.00539-06
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发表时间：
2006-07-01
影响因子：
4.4
通讯作者：
Legisa, Matic
中科院分区：
文献类型：
--
作者：
Mlakar, Tina;Legisa, Matic
尼日尔6-磷酸果糖-1-激酶（PFK 1）有两种形式，85 kDa的天然酶和49 kDa的较短片段，前者通过翻译后修饰形成。到目前为止，动力学特性从来没有被确定的酶纯化到接近同质。对于第一次，动力学参数测定个别酶柠檬酸盐抑制。天然的85-kDa的酶被认为是适度抑制柠檬酸盐，Ki值确定为1.5 mM，在系统中与5 mM Mg 2+离子，而镁浓度增加缓解柠檬酸盐的负面影响。一个相同的抑制系数也确定在铵离子的存在下，虽然铵作为一个强的酶活性的激活剂。另一方面，PFK 1的较短片段被证明对柠檬酸盐的抑制具有完全抗性。变构柠檬酸盐结合位点最有可能在天然蛋白质的C-末端部分截短后丢失，已知抑制剂的一些结合位点位于该区域。在接近生理条件下，其特征在于低果糖-6-磷酸浓度，在体外实验期间观察到更高效率的较短片段。由于该酶对特异性配体的阳性对照变得更敏感，而阴性对照在翻译后修饰后丢失，因此较短的PFK 1片段似乎是最负责通过A中的糖酵解产生不受干扰的代谢流的酶。尼日尔细胞
Two forms of Aspergillus niger 6-phosphofructo-1-kinase (PFK1) have been described recently, the 85-kDa native enzyme and 49-kDa shorter fragment that is formed from the former by posttranslational modification. So far, kinetic characteristics have never been determined on the enzyme purified to near homogeneity. For the first time, kinetic parameters were determined for individual enzymes with respect to citrate inhibition. The native 85-kDa enzyme was found to be moderately inhibited by citrate, with the Ki value determined to be 1.5 mM, in the system with 5 mM Mg2+ ions, while increasing magnesium concentrations relieved the negative effect of citrate. An identical inhibition coefficient was determined also in the presence of ammonium ions, although ammonium acted as a strong activator of enzyme activity. On the other hand, the shorter fragment of PFK1 proved to be completely resistant to inhibition by citrate. Allosteric citrate binding sites were most probably lost after the truncation of the C-terminal part of the native protein, in which region some binding sites for inhibitor are known to be located. At near physiological conditions, characterized by low fructose-6-phosphate concentrations, a much higher efficiency of the shorter fragment was observed during an in vitro experiment. Since the enzyme became more susceptible to the positive control by specific ligands, while the negative control was lost after posttranslational modification, the shorter PFK1 fragment seems to be the enzyme most responsible for generating undisturbed metabolic flow through glycolysis in A. niger cells.