beta-deuterium kinetic isotope effects in the purine nucleoside phosphorylase reaction.
beta-deuterium kinetic isotope effects in the purine nucleoside phosphorylase reaction.
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β-氘动力学同位素对嘌呤核苷磷酸化酶反应的影响。
DOI：
10.1042/bj2780487
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Krenitsky,TA
中科院分区：
文献类型：
--
作者：
Guo,XM;Ashwell,M;Sinnott,ML;Krenitsky,TA
1.以肌苷为原料，经3′和5′位的四异丙基二氨酰保护，二甲基亚砜和乙酸酐氧化，NaB 2 H4立即还原羰基糖产物，保护的[2 ′-2H]阿拉伯肌苷经三氟甲磺酰化转化为C-2′位，与丙酸铯反应，脱保护，得到[2 ′-2H]肌苷。2.平衡扰动技术被用来测量β 2 H（V/K）的磷酸化的这种化合物的嘌呤核苷磷酸化酶的大肠杆菌作为pH值的函数。pH值的变化表明，1.068的内在效应被最佳pH值附近的同位素沉默步骤所掩盖。4.先前测量的这些β-氘效应和α-氘效应的类似pH变化[Stein & Cordes（1981）J. Biol. Chem. 256，767-772; Lehikoinen，Sinnott和Krenitsky（1989）Biochem.J.257，355-359]因为这种反应提供了第一个实验保证的共同假设，pH值的变化只是掩盖和揭露化学步骤，酶催化的反应，并且不可检测地改变过渡态结构。5.过渡态的C-H-2′键和缺电子p轨道之间的二面角在32-48度的范围内，与基本上平面的呋喃糖环雅阁。
1. [2′-2H]Inosine was made from inosine by tetraisopropyldisiloxanyl protection of the 3′- and 5′-positions, oxidation with dimethyl sulphoxide and acetic anhydride, immediate NaB2H4 reduction of the oxo sugar product and inversion at C-2′ of the resultant protected [2′-2H]arabino-inosine by trifluoromethanesulphonylation and reaction with caesium propionate, followed by deprotection. 2. The equilibrium-perturbation technique was used to measure beta 2H(V/K) for phosphorolysis of this compound by the purine nucleoside phosphorylase of Escherichia coli as a function of pH. 3. The pH variation indicates an intrinsic effect of 1.068 masked by isotopically silent steps near the pH optimum. 4. The similar pH variation of these beta-deuterium effects and the alpha-deuterium effects measured previously [Stein & Cordes (1981) J. Biol. Chem. 256, 767-772; Lehikoinen, Sinnott & Krenitsky (1989) Biochem. J. 257, 355-359] for this reaction provides the first experimental reassurance for the common assumption that pH changes merely mask and unmask the chemical steps in an enzyme-catalysed reaction, and do not detectably alter transition-state structure. 5. The dihedral angle between the C-H-2′ bond and the electron-deficient p-orbital at the transition state is in the range 32-48 degrees, in accord with an essentially planar furanose ring.