Kinukawa Masashi, Vacquier Victor D
Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA 92093-0202 USA.
J Biochem. 2007 Oct;142(4):501-6. doi: 10.1093/jb/mvm154. Epub 2007 Aug 30.
Adenylate kinase (AK) is localized in sea urchin sperm flagella and embryonic cilia. To investigate sea urchin Strongylocentrotus purpuratus AK (SpAK) enzymatic characteristics, the full-length recombinant protein of 130 kDa (SpAKr) and each of its three catalytic domains were expressed in Escherichia coli. Although the full-length SpAK had high enzymatic activity, each of the three catalytic domains had no activity. The Km for ATP synthesis from ADP was 0.23 mM and the Vmax was 4.51 mumol ATP formed per minute per milligram of protein. The specific AK inhibitor, Ap5A, blocks SpAKr enzymatic activity with an IC50 of 0.53 microM. The pH optimum for SpAKr is 8.1, as compared to 7.7 for the natural SpAK. Calcium inhibits SpAKr activity in a dose-dependent manner. Although SpAKr has three cAMP-dependent protein kinase phosphorylation sites, and can be phosphorylated in vitro, the enzymatic kinetics after phosphorylation are not significantly altered. SpAK and Chlamydomonas flagellar AKs are the only AKs with three catalytic sites. Further study of the SpAKr will aid in understanding the active site of this interesting and important ATP synthase.
腺苷酸激酶（AK）定位于海胆精子鞭毛和胚胎纤毛中。为了研究海胆紫海胆AK（SpAK）的酶学特性，在大肠杆菌中表达了130 kDa的全长重组蛋白（SpAKr）及其三个催化结构域。尽管全长SpAK具有较高的酶活性，但三个催化结构域均无活性。由ADP合成ATP的Km为0.23 mM，Vmax为每毫克蛋白质每分钟形成4.51 μmol ATP。特异性AK抑制剂Ap5A以0.53 μM的IC50阻断SpAKr的酶活性。SpAKr的最适pH为8.1，而天然SpAK的最适pH为7.7。钙以剂量依赖的方式抑制SpAKr的活性。尽管SpAKr有三个依赖于cAMP的蛋白激酶磷酸化位点，并且可以在体外被磷酸化，但磷酸化后的酶动力学没有显著改变。SpAK和衣藻鞭毛AK是仅有的具有三个催化位点的AK。对SpAKr的进一步研究将有助于理解这种有趣且重要的ATP合酶的活性位点。
