Liu Lu, Cottrell Joseph W, Scott Lincoln G, Fedor Martha J
Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA.
Nat Chem Biol. 2009 May;5(5):351-7. doi: 10.1038/nchembio.156. Epub 2009 Mar 29.
Active site guanines are critical for self-cleavage reactions of several ribozymes, but their precise functions in catalysis are unclear. To learn whether protonated or deprotonated forms of guanine predominate in the active site, microscopic pKa values were determined for ionization of 8-azaguanosine substituted for G8 in the active site of a fully functional hairpin ribozyme in order to determine microscopic pKa values for 8-azaguanine deprotonation from the pH dependence of fluorescence. Microscopic pKa values above 9 for deprotonation of 8-azaguanine in the active site were about 3 units higher than apparent pKa values determined from the pH dependence of self-cleavage kinetics. Thus, the increase in activity with increasing pH does not correlate with deprotonation of G8, and most of G8 is protonated at neutral pH. These results do not exclude a role in proton transfer, but a simple interpretation is that G8 functions in the protonated form, perhaps by donating hydrogen bonds.
活性位点鸟嘌呤对于几种核酶的自我切割反应至关重要，但其在催化过程中的精确功能尚不清楚。为了了解鸟嘌呤的质子化或去质子化形式在活性位点是否占主导，我们测定了在完全功能性发夹状核酶的活性位点中，替代G8的8-氮杂鸟苷电离的微观pKa值，以便从荧光的pH依赖性确定8-氮杂鸟嘌呤去质子化的微观pKa值。活性位点中8-氮杂鸟嘌呤去质子化的微观pKa值高于9，比根据自我切割动力学的pH依赖性确定的表观pKa值高约3个单位。因此，活性随pH升高而增加与G8的去质子化无关，并且在中性pH下大多数G8是质子化的。这些结果不排除在质子转移中起作用，但一个简单的解释是G8以质子化形式起作用，可能是通过提供氢键。
