Kati W M, Wolfenden R
Department of Biochemistry, University of North Carolina, Chapel Hill 27514.
Biochemistry. 1989 Sep 19;28(19):7919-27. doi: 10.1021/bi00445a055.
Adenosine deaminase was found to bind 6-hydroxy-1,6-dihydropurine ribonucleoside (II), formed by reversible addition of water to purine ribonucleoside (I) in a reaction analogous to formation of a tetrahedral intermediate in substrate deamination, with an apparent Ki value of 3 x 10(-13) M at 20 degrees C. 1,6-Dihydropurine ribonucleoside (IV), synthesized by photolysis of purine ribonucleoside in the presence of NaBH4, exhibited a Ki value of 5.4 x 10-6 M. After correction for differences between the relative free energies of solvation of II and IV, the 6-hydroxyl group of II was estimated to contribute more than 16 kcal to the free energy of binding, approaching the enthalapy of formation of a single hydrogen bond to charged group in the vapor phase. The relatively weak binding of IV and of substrate water suggests that entropic effects, arising from the cooperative action of binding determinants contained within these separate molecules, contribute more than 10 kcal/mol to the free energy of binding of II in which these binding determinants are contained within a single molecule. In free solution, the entropy of reversible hydration of I was evaluated by measuring the temperature dependence of equilibria of protonation of I and of pseudobase formation from I-methylpurinium ribonucleoside as -35 eu, comparable with the entropy of activation for the uncatalyzed hydrolysis of adenosine. In the active site of adenosine deaminase, this thermodynamic obstacle is evidently climbed spontaneously as a result of attractive interactions between the active site and the critical hydroxyl group at the 6-position.(ABSTRACT TRUNCATED AT 250 WORDS)
腺苷脱氨酶被发现可结合6-羟基-1,6-二氢嘌呤核糖核苷（II），它是由嘌呤核糖核苷（I）通过与底物脱氨反应中四面体中间体形成类似的反应可逆加水形成的，在20℃时其表观解离常数（Ki）值为3×10⁻¹³ M。通过在NaBH₄存在下光解嘌呤核糖核苷合成的1,6-二氢嘌呤核糖核苷（IV），其Ki值为5.4×10⁻⁶ M。在对II和IV溶剂化相对自由能差异进行校正后，估计II的6-羟基对结合自由能的贡献超过16千卡，接近气相中与带电基团形成单个氢键的焓。IV与底物水的相对较弱结合表明，这些单独分子中所含结合决定簇的协同作用产生的熵效应，对包含这些结合决定簇的单个分子II的结合自由能贡献超过10千卡/摩尔。在自由溶液中，通过测量I质子化平衡以及I-甲基嘌呤核糖核苷假碱形成平衡的温度依赖性，评估I可逆水合的熵为-35熵单位，与腺苷未催化水解的活化熵相当。在腺苷脱氨酶的活性位点，由于活性位点与6位关键羟基之间的吸引相互作用，这个热力学障碍显然被自发跨越。（摘要截短于250字）
