College of Life Science, Hubei Normal University, Huangshi, Hubei 435002, PR China.
Int J Biol Macromol. 2011 Jul 1;49(1):98-102. doi: 10.1016/j.ijbiomac.2011.04.002. Epub 2011 Apr 12.
Arginine kinase (AK), a crucial enzyme in energy metabolism, buffers cellular ATP levels by catalyzing the reversible phosphoryl transfer between ATP and arginine. To better understand the role of Cys271 in conformational changes of AK from greasyback shrimp (Metapenaeus ensis), we replaced the residue with serine and alanine. A detailed comparison of the catalytic activity and conformation was made between wild-type AK and the mutants by means of activity analysis, ultraviolet (UV) difference, fluorescence spectrum and size exclusion chromatography (SEC). The results indicated that the catalytic activity of the two mutants was gone. The substrates, arginine-ADP-Mg(2+) could induce conformational changes, and additional NO(3)(-) could induce further changes in both the native enzyme and the variants. We speculated that Cys271 might be located in the hinge region between the two domains of AK and cause enzyme conformational changes upon addition of substrate.
精氨酸激酶（AK）是能量代谢中的关键酶，通过催化 ATP 和精氨酸之间的可逆磷酸转移来缓冲细胞内的 ATP 水平。为了更好地了解半滑舌鳎 AK 中 Cys271 残基在构象变化中的作用，我们用丝氨酸和丙氨酸替换了该残基。通过活性分析、紫外（UV）差谱、荧光光谱和尺寸排阻色谱（SEC）对野生型 AK 和突变体的催化活性和构象进行了详细比较。结果表明，两个突变体的催化活性丧失。底物精氨酸-ADP-Mg(2+)可诱导构象变化，外加的 NO(3)(-)可引起天然酶和变体的进一步变化。我们推测 Cys271 可能位于 AK 两个结构域之间的铰链区域，在底物加入时引起酶的构象变化。
