Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska Institute, F68, SE-141 86 Huddinge, Sweden.
Int J Biochem Cell Biol. 2013 May;45(5):925-31. doi: 10.1016/j.biocel.2013.02.004. Epub 2013 Feb 14.
Adenylate kinases regulate adenine nucleotide levels and are present in different intracellular compartments. These enzymes also participate in the activation of pharmacologically active nucleoside and nucleotide analogs. We have in the present study identified the ninth isoform of the adenylate kinase family of enzymes and accordingly named the protein adenylate kinase 9 (AK9). Initially a full-length cDNA of a hypothetical protein containing a predicted adenylate kinase domain was identified and subsequently cloned and expressed in Escherichia coli. The substrate specificity of the recombinant protein showed that the enzyme catalyzed the phosphorylation of AMP, dAMP, CMP and dCMP with ATP as phosphate donor, while only AMP and CMP were phosphorylated when GTP was the phosphate donor. The kinetic parameters of AK9 were determined for AMP, dAMP and CMP with ATP as phosphate donor. Interestingly, in addition to the diphosphate products, a nucleoside diphosphate kinase (NDPK) activity was also present with subsequent triphosphates formed. With ATP or GTP as phosphate donor it was possible to detect the production of ATP, CTP, GTP, UTP, dATP, dCTP, dGTP and TTP as enzymatic products from the corresponding diphosphate substrates. A number of previously characterized adenylate kinases were also tested and found to possess a broad phosphotransferase activity similar to AK9. These enzymes are accordingly suggested to be regarded as nucleoside mono- and diphosphate kinases with catalytic activities possibly determined by local substrate concentrations.
腺嘌呤激酶调节腺嘌呤核苷酸水平，并存在于不同的细胞内隔室中。这些酶还参与药理学活性核苷和核苷酸类似物的激活。在本研究中，我们鉴定了腺嘌呤激酶家族的第九种同工酶，因此将该蛋白命名为腺嘌呤激酶 9（AK9）。最初，鉴定了一个包含预测腺嘌呤激酶结构域的假设蛋白的全长 cDNA，随后在大肠杆菌中进行克隆和表达。重组蛋白的底物特异性表明，该酶催化 AMP、dAMP、CMP 和 dCMP 的磷酸化，而当 GTP 为磷酸供体时，仅 AMP 和 CMP 被磷酸化。使用 ATP 作为磷酸供体时，测定了 AK9 的 AMP、dAMP 和 CMP 的动力学参数。有趣的是，除了二磷酸产物外，还存在核苷二磷酸激酶（NDPK）活性，随后形成三磷酸。使用 ATP 或 GTP 作为磷酸供体，有可能从相应的二磷酸底物中检测到 ATP、CTP、GTP、UTP、dATP、dCTP、dGTP 和 TTP 的产生作为酶产物。还测试了一些先前表征的腺嘌呤激酶，发现它们具有类似 AK9 的广泛磷酸转移酶活性。因此，这些酶被建议被视为核苷单磷酸和二磷酸激酶，其催化活性可能由局部底物浓度决定。
