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首页> 外文期刊>Biochemistry >Pentavlanet Ions Dependecy Is a Conserved Property of Adenosine Kinase from Diverse Sources: Identificationo of a Novel Motif Implicated in Phosphate and Magnesium Ion Binding and Substrate Inhibition
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Pentavlanet Ions Dependecy Is a Conserved Property of Adenosine Kinase from Diverse Sources: Identificationo of a Novel Motif Implicated in Phosphate and Magnesium Ion Binding and Substrate Inhibition

机译：单价离子依赖性是来自多种来源的腺苷激酶的保守性质：涉及磷酸盐和镁离子结合和底物抑制的新型母题的鉴定。

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摘要

The catalytic activity of adenosine kinase (AK) from mammalian sources has previously been shown to exhibit a marked dependency upon the presence of pentavalent ions (PVI), such as phosphate (PO_4), arsenate, or vanadate. We now show that the activity of AK from diverse sources, including plant, yeast, and protist species, is also markedly enhanced in the presence of PVI. In all cases, PO_4 or other PVI exerted their effects primarily by decreasing the K_m for adenosine and alleviating the inhibition caused by high concentrations of substrate. These results provide evidence that PVI dependency is a conserved property of AK and perhaps of the PfkB family of carbohydrate kinases which includes AK. On the basis of sequence alginments, we have identified a conserved motif NXXE within the PfkB family. The N and E of this motif make close contacts with Mg~(2+) and PO_4 ions in the crystal strucutres of AK and bacterial ribokinase (another PfkB member which shows PVI dependency), implicating these residues in their binding. Site-directed mutagenesis of these residues in Chinese hamster AK have resulted in active proteins with greatly altered phosphate stimulation and substrate inhibition characteristics. The N239Q mutation leads to the formation of an active protien whose activity was not stimulated by PO_4 or inhibited by high concentrations of adenosine or ATP. The activity of the E242D mutant protein was also not significantly altered in the presence of phosphate. Although PO_4 had no effect on the K_m~(Adenosine) for this mutant, the K_m~(ATP), K_i~(Adenosine), and K_i~(ATP) were signficantly decreased. In contrast to these mutations, N239L or E242L mutant proteins showed greatly decreased activity with an altered Mg~(2+) requirement. These observations support the view that N239 and E242 play an important role in the binding of PO_4 and Mg~(2+) ions required for the catalytic activity of adenosine kinase.
机译：先前已显示来自哺乳动物来源的腺苷激酶（AK）的催化活性对五价离子（PVI）（例如磷酸根（PO_4），砷酸根或钒酸根）的存在表现出明显的依赖性。我们现在显示，在存在PVI的情况下，来自植物，酵母和原生生物等多种来源的AK的活性也显着增强。在所有情况下，PO_4或其他PVI主要通过降低腺苷的K_m并减轻高浓度底物引起的抑制来发挥作用。这些结果提供了证据，证明PVI依赖性是AK的保守性质，并且可能是包括AK的碳水化合物激酶的PfkB家族的保守性质。根据序列alginations，我们已经确定了PfkB家族内的保守基序NXXE。该基序的N和E与AK和细菌核糖激酶（另一个显示PVI依赖性的PfkB成员）的晶体结构中的Mg〜（2+）和PO_4离子紧密接触，这意味着这些残基的结合。这些残基在中国仓鼠AK中的定点诱变产生了活性蛋白，其磷酸盐刺激和底物抑制特性大大改变。 N239Q突变导致活性蛋白的形成，其活性不受PO_4刺激或未被高浓度的腺苷或ATP抑制。在磷酸盐的存在下，E242D突变蛋白的活性也没有明显改变。尽管PO_4对该突变体的K_m〜（腺苷）没有影响，但是K_m〜（ATP），K_i〜（腺苷）和K_i〜（ATP）显着降低。与这些突变相反，N239L或E242L突变蛋白在Mg〜（2+）需求改变的情况下，其活性大大降低。这些观察结果支持以下观点：N239和E242在腺苷激酶催化活性所需的PO_4和Mg〜（2+）离子的结合中起重要作用。

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