Maj Mary C, Singh Bhag, Gupta Radhey S
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada, L8N 3Z5.
Biochemistry. 2002 Mar 26;41(12):4059-69. doi: 10.1021/bi0119161.
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 (PO4), 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, PO4 or other PVI exerted their effects primarily by decreasing the Km for adenosine and alleviating the inhibition caused by high concentrations of substrates. 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 alignments, we have identified a conserved motif NXXE within the PfkB family. The N and E of this motif make close contacts with Mg2+ and PO4 ions in the crystal structures 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 protein whose activity was not stimulated by PO4 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 PO4 had no effect on the KmAdenosine for this mutant, the KmATP, K(i)Adenosine, and K(i)ATP were significantly decreased. In contrast to these mutations, N239L or E242L mutant proteins showed greatly decreased activity with an altered Mg2+ requirement. These observations support the view that N239 and E242 play an important role in the binding of PO4 and Mg2+ ions required for the catalytic activity of adenosine kinase.
先前已表明，来自哺乳动物的腺苷激酶（AK）的催化活性对五价离子（PVI）如磷酸盐（PO4）、砷酸盐或钒酸盐的存在表现出显著依赖性。我们现在表明，包括植物、酵母和原生生物物种在内的多种来源的AK活性在PVI存在时也显著增强。在所有情况下，PO4或其他PVI主要通过降低腺苷的Km并减轻高浓度底物引起的抑制作用来发挥其效应。这些结果证明PVI依赖性是AK以及可能包括AK的PfkB家族碳水化合物激酶的保守特性。基于序列比对，我们在PfkB家族中鉴定出一个保守基序NXXE。该基序的N和E在AK和细菌核糖激酶（另一个显示PVI依赖性的PfkB成员）的晶体结构中与Mg2+和PO4离子紧密接触，表明这些残基参与它们的结合。对中国仓鼠AK中这些残基进行定点诱变产生了具有极大改变的磷酸盐刺激和底物抑制特性的活性蛋白。N239Q突变导致形成一种活性蛋白，其活性不受PO4刺激，也不受高浓度腺苷或ATP抑制。在磷酸盐存在下，E242D突变蛋白的活性也没有显著改变。尽管PO4对该突变体的Km腺苷没有影响，但KmATP、K（i）腺苷和K（i）ATP显著降低。与这些突变相反，N239L或E242L突变蛋白的活性大大降低，且对Mg2+的需求发生改变。这些观察结果支持这样的观点，即N239和E242在腺苷激酶催化活性所需的PO4和Mg2+离子结合中起重要作用。
