Lupták A, Ferré-D'Amaré A R, Zhou K, Zilm K W, Doudna J A
Department of Chemistry, Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520, USA.
J Am Chem Soc. 2001 Sep 5;123(35):8447-52. doi: 10.1021/ja016091x.
Hepatitis delta virus ribozymes have been proposed to perform self-cleavage via a general acid/base mechanism involving an active-site cytosine, based on evidence from both a crystal structure of the cleavage product and kinetic measurements. To determine whether this cytosine (C75) in the genomic ribozyme has an altered pK(a) consistent with its role as a general acid or base, we used (13)C NMR to determine its microscopic pK(a) in the product form of the ribozyme. The measured pK(a) is moderately shifted from that of a free nucleoside or a base-paired cytosine and has the same divalent metal ion dependence as the apparent reaction pK(a)'s measured kinetically. However, under all conditions tested, the microscopic pK(a) is lower than the apparent reaction pK(a), supporting a model in which C75 is deprotonated in the product form of the ribozyme at physiological pH. While additional results suggest that the pK(a) is not shifted in the reactant state of the ribozyme, these data cannot rule out elevation of the C75 pK(a) in an intermediate state of the transesterification reaction.
基于裂解产物晶体结构和动力学测量的证据，有人提出丁型肝炎病毒核酶通过涉及活性位点胞嘧啶的一般酸碱机制进行自我切割。为了确定基因组核酶中的这种胞嘧啶（C75）是否具有与其作为一般酸或碱的作用相一致的改变的pK(a)，我们使用(13)C NMR来确定其在核酶产物形式中的微观pK(a)。测得的pK(a)与游离核苷或碱基配对胞嘧啶的pK(a)有适度偏移，并且与动力学测量的表观反应pK(a)具有相同的二价金属离子依赖性。然而，在所有测试条件下，微观pK(a)低于表观反应pK(a)，这支持了一种模型，即在生理pH下，C75在核酶产物形式中去质子化。虽然其他结果表明在核酶的反应物状态下pK(a)没有偏移，但这些数据不能排除在酯交换反应的中间状态下C75 pK(a)升高的可能性。
