Chang G G, Shiao M S, Lee K R, Wu J J
Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Biochem J. 1990 Dec 15;272(3):683-90. doi: 10.1042/bj2720683.
Oxidation of 1,N6-ethenoadenosine monophosphate (epsilon AMP) with periodate cleaved the cis-diol of the ribose ring and resulted in the formation of a dialdehyde derivative (epsilon AMP-dial). At room temperature epsilon AMP-dial was unstable and underwent beta-elimination to give 4',5'-anhydro-1,N6-ethenoadenosine dialdehyde acetal (A epsilon Ado-dial). These nucleotide analogues were found to inactivate human placental alkaline phosphatase in a time- and concentration-dependent manner. epsilon AMP-dial was shown to be an affinity label for the enzyme on the basis of the following criteria. (a) Kinetics of the enzyme activity loss over a wide range of epsilon AMP-dial concentration showed a saturating phenomenon. Removal of the phosphate group made the reagent (A epsilon Ado-dial) become a general chemical modifying reagent. (b) The artificial substrate p-nitrophenyl phosphate gave substantial protection of the enzyme against inactivation. (c) epsilon AMP-dial was a substrate and a partial mixed-type inhibitor for the enzyme. Results of the inhibition and protection studies indicated that the reagent and substrate could combine with the enzyme simultaneously. Besides the phosphate-binding domain, an induced hydrophobic region is proposed for the substrate-binding site for human placental alkaline phosphatase.
用高碘酸盐氧化1,N6-乙烯腺苷单磷酸（ε-AMP）可裂解核糖环的顺式二醇，生成二醛衍生物（ε-AMP-二醛）。在室温下，ε-AMP-二醛不稳定，会发生β-消除反应，生成4',5'-脱水-1,N6-乙烯腺苷二醛缩醛（AεAdo-二醛）。发现这些核苷酸类似物能以时间和浓度依赖性方式使胎盘碱性磷酸酶失活。基于以下标准，ε-AMP-二醛被证明是该酶的亲和标记物。（a）在宽范围的ε-AMP-二醛浓度下酶活性丧失的动力学表现出饱和现象。去除磷酸基团使试剂（AεAdo-二醛）成为一种通用的化学修饰试剂。（b）人工底物对硝基苯磷酸对酶失活有显著的保护作用。（c）ε-AMP-二醛是该酶的底物和部分混合型抑制剂。抑制和保护研究结果表明，该试剂和底物可同时与酶结合。除了磷酸结合结构域，还提出了一个诱导的疏水区域作为胎盘碱性磷酸酶底物结合位点。
