Direct measurement of a pKa near neutrality for the catalytic cytosine in the genomic HDV ribozyme using Raman crystallography
Direct measurement of a pKa near neutrality for the catalytic cytosine in the genomic HDV ribozyme using Raman crystallography
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DOI：
10.1021/ja0743893
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发表时间：
2007-10-31
影响因子：
15
通讯作者：
Carey, Paul R.
中科院分区：
文献类型：
--
作者：
Gong, Bo;Chen, Jui-Hui;Carey, Paul R.
丁型肝炎病毒（HDV）核酶使用胞嘧啶来促进一般的酸碱催化。生物化学研究表明，C75的pK（a）被扰动至接近中性。为了直接测量该pK（a），使用拉曼显微镜在单个核酶晶体上记录拉曼光谱。在1528 cm（-1）处发现了一个中性胞嘧啶的光谱特征。在低pH下，该模式被新的光谱特征所取代。监测这些特征作为pH的函数，揭示了与Mg 2+结合反协同偶联的胞嘧啶的pK（a）值，在20和2 MM Mg 2+存在下的值分别为6.15和6.40。这些pK（a）值与从溶液中的核酶活性实验获得的值吻合得很好。为了将观察到的pK（a）与特定的核苷酸相关联，检查了无催化活性的C75 U晶体。拉曼差谱表明，这种突变并不影响核酶的构象。然而，晶体的C75 U没有产生一个信号，从质子化胞嘧啶，提供了强有力的证据表明，质子化的C75正在监测野生型核酶。这些研究提供了核酶中催化残基接近中性的pK（a）的第一个直接物理测量，并表明核酶与其蛋白酶对应物一样，可以优化其侧链的pK（a）以进行质子转移。
The hepatitis delta virus (HDV) ribozyme uses a cytosine to facilitate general acid-base catalysis. Biochemical studies suggest that C75 has a pK(a) perturbed to near neutrality. To measure this pK(a) directly, Raman spectra were recorded on single ribozyme crystals using a Raman microscope. A spectral feature arising from a single neutral cytosine was identified at 1528 cm(-1). At low pH, this mode was replaced with a new spectral feature. Monitoring these features as a function of pH revealed pK(a) values for the cytosine that couple anti cooperatively with Mg2+ binding, with values of 6.15 and 6.40 in the presence of 20 and 2 MM Mg2+, respectively. These pK(a) values agree well with those obtained from ribozyme activity experiments in solution. To correlate the observed pK(a) with a specific nucleotide, crystals of C75U, which is catalytically inactive, were examined. The Raman difference spectra show that this mutation does not affect the conformation of the ribozyme. However, crystals of C75U did not produce a signal from a protonatable cytosine, providing strong evidence that protonation of C75 is being monitored in the wild-type ribozyme. These studies provide the first direct physical measurement of a pK(a) near neutrality for a catalytic residue in a ribozyme and show that ribozymes, like their protein enzyme counterparts, can optimize the pK(a) of their side chains for proton transfer.