Morange M, Blanco F G, Vandenbunder B, Buc H
Eur J Biochem. 1976 Jun 1;65(2):553-63. doi: 10.1111/j.1432-1033.1976.tb10373.x.
A series of AMP analogs has been selected in order to better understand the structural requirements (a) for the efficient binding of the activator molecule at the correct site on phosphorylase b from rabbit skeletal muscle and (b) for the activation which is observed. Two types of activation are known, according to Black and Wang [J. Biol. Chem. 243, 5892-5898 (1968)]: either a cooperative response with respect to the activator concentration (like the one which is obtained for AMP itself) or a non-cooperative response observed in the case of IMP. It is shown that the 5'-phosphate moiety is absolutely required for the analog to bind at the correct site (adenine or adenosine bind at another enzymic site), and that the free enthalpy, delta G, corresponding to the association process varies in a complex manner with respect to the substitution of the different positions of the AMP molecule. Moreover, the differences delta G (analog) - delta G (AMP) = delta G obtained for two types of substitution separately do not add up to the same energy difference as the one obtained when the two substitutions are made simultaneously on the AMP molecule. It appears that all the mononucleotides which have been tested up to now may be divided into two classes. Class I (AMP class) is characterized, apart from a strong activation, by the following features: (a) one molecule of analog expels two molecules of bound glucose 6-phosphate as it binds on the enzyme; (b) bound analog protects slowly one crucial cysteinyl residue against attack by 5,5'-dithio-bis(2-nitrobenzoic acid) at 4 degrees C; (c) association of two molecules of dimer is strengthened at 4 degrees C in the presence of the analog. Class II (IMP class) is associated with a weak activation and with the following set of properties: (a) a single molecule of bound glucose 6-phosphate is released as the first molecule of analog binds on the dimer; (b) two slowly reacting cysteinyl residues per subunit are immediately protected against 5,5'-dithio-bis(2-nitrobenzoic acid) by the binding of the analog at 4 degrees C; (c) the analog dissociates the low amount of tetramer which is present at 4 degrees C in the absence of AMP into two molecules of dimer. These results are discussed according to a plausible scheme of transconformations taking place in glycogen phosphorylase b, a model which has been derived earlier by relaxation studies.
为了更好地理解（a）激活剂分子在兔骨骼肌磷酸化酶b的正确位点高效结合的结构要求，以及（b）所观察到的激活作用的结构要求，已选择了一系列AMP类似物。根据布莱克和王[《生物化学杂志》243, 5892 - 5898 (1968)]的研究，已知有两种类型的激活：要么是对激活剂浓度的协同反应（类似于AMP自身的反应），要么是在IMP情况下观察到的非协同反应。结果表明，5'-磷酸部分是类似物在正确位点结合所绝对必需的（腺嘌呤或腺苷在另一个酶位点结合），并且与缔合过程相对应的自由焓ΔG随AMP分子不同位置的取代而以复杂的方式变化。此外，分别对两种类型取代获得的ΔG（类似物） - ΔG（AMP） = ΔG的差异，与在AMP分子上同时进行两种取代时获得的能量差异并不相同。似乎到目前为止所测试的所有单核苷酸都可分为两类。I类（AMP类）的特征除了具有强烈的激活作用外，还具有以下特点：（a）一个类似物分子在与酶结合时会排出两个结合的葡萄糖6-磷酸分子；（b）结合的类似物在4℃时缓慢保护一个关键的半胱氨酰残基免受5,5'-二硫代双（2-硝基苯甲酸）的攻击；（c）在类似物存在下，4℃时二聚体的两个分子的缔合得到加强。II类（IMP类）与弱激活以及以下一组性质相关：（a）当第一个类似物分子与二聚体结合时，会释放一个结合的葡萄糖6-磷酸分子；（b）类似物在4℃结合时，每个亚基的两个反应缓慢半胱氨酰残基会立即受到保护，免受5,5'-二硫代双（2-硝基苯甲酸）的攻击；（c）类似物会使在4℃时不存在AMP的情况下存在的少量四聚体解离成两个二聚体分子。根据糖原磷酸化酶b中发生的一种可能的转构象方案对这些结果进行了讨论，该模型是先前通过弛豫研究得出的。
