Yakovlev G I, Moiseyev G P
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow.
Biochim Biophys Acta. 1993 Sep 3;1202(1):143-8. doi: 10.1016/0167-4838(93)90075-3.
The chemical-shift dependences of the proton signals of the guanosine and uridine moieties were measured as a function of the relative amount of GpcU complexed with RNase Pb1 (EC 3.1.27.3). The equal values of the chemical-shift changes of the guanosine C8-protons on complex formation between GpcU and RNase Pb1 and that of the 3'-GMP and RNase Pb1 allow to conclude that the guanosine base is bound in the same manner in these protein-ligand complexes. The guanosine moiety of GpcU is also most probably bound in the syn-conformation. The absence of changes in both the linewidths and the chemical shifts of the C1', C5 and C6-proton signals of the uridine on complex formation indicates that the uridine moiety of the dinucleoside phosphonate is not immobilized in the complex. The pH dependences of the chemical shifts of the C2-protons of the histidine-imidazole ring of RNase Pb1 and that of the 31P of GpcU in the RNase complex were studied. The results suggest that there is a direct interaction between the phosphonate group of the ligand and the protonated imidazole ring of His-90. The side groups of His-38 and Glu-56 are hydrogen bonded to each other at neutral pH and they are located in the vicinity of the phosphonate group of GpcU. When the carboxyl group of Glu-56 is protonated the His-38 imidazole ring forms a new hydrogen bond with one of the phosphoryl oxygens of the phosphonate group. On the basis of these results we propose the mechanism of action of RNase Pb1 which is probably also true for RNase T1.
测量了鸟苷和尿苷部分质子信号的化学位移依赖性，其作为与核糖核酸酶Pb1（EC 3.1.27.3）复合的GpcU相对量的函数。GpcU与核糖核酸酶Pb1形成复合物时鸟苷C8-质子化学位移变化的值，与3'-GMP和核糖核酸酶Pb1形成复合物时的相等，这使得可以得出结论，在这些蛋白质-配体复合物中鸟苷碱基以相同方式结合。GpcU的鸟苷部分很可能也以顺式构象结合。复合物形成时尿苷的C1'、C5和C6-质子信号的线宽和化学位移均无变化，这表明二核苷膦酸酯的尿苷部分在复合物中未被固定。研究了核糖核酸酶Pb1的组氨酸-咪唑环C2-质子以及核糖核酸酶复合物中GpcU的31P化学位移的pH依赖性。结果表明，配体的膦酸酯基团与His-90的质子化咪唑环之间存在直接相互作用。His-38和Glu-56的侧链在中性pH下相互氢键结合，且它们位于GpcU膦酸酯基团附近。当Glu-56的羧基质子化时，His-38咪唑环与膦酸酯基团的一个磷酰氧形成新的氢键。基于这些结果，我们提出了核糖核酸酶Pb1的作用机制，这可能对核糖核酸酶T1也适用。
