PSEUDOURIDINE SYNTHASES

假尿苷合成酶存在于所有生物体中，催化RNA中特定的尿苷残基异构化为假尿苷。伪尿苷的特定生理作用最近也出现了：1)伪尿苷在U2 SnRNA中是必需的，以功能组装U2 SnRNP，剪接Pre-mRNA；2)先天性角化不良是由于缺乏与伪尿苷合成酶TRUB序列相似的蛋白质引起的；3)伪尿苷合成酶的抑制部分解释了5-氟尿嘧啶的细胞毒性，5-氟尿嘧啶是一种抗癌药物，可转化为5-氟尿嘧啶并在体内并入RNA。对两种假尿苷合成酶--TRUB和RluA进行了机理研究。活性部位的事件将通过整合定点突变、化学修饰、动力学同位素效应的测定以及使用核磁共振方法直接观察假性尿苷合成酶与含5-氟尿苷的RNA之间的加合物来阐明。定点突变揭示了天冬氨酸的催化作用，而定点化学修饰将探索该关键天冬氨酸所起的催化作用。动力学同位素效应将描绘出受该氨基酸影响的反应步骤(S)。将测定含有5-氟尿苷的RNA与突变蛋白之间加合物的化学结构，并与野生型酶进行比较。这种比较将通过识别蛋白质和RNA中参与相互作用的特定基团来揭示蛋白质-RNA相互作用的性质，这对于理解伪尿苷合成酶催化和5-氟尿嘧啶的作用是至关重要的。这些研究将扩大到检查更多与催化有关的氨基酸残基，包括半胱氨酸残基和TRUB的氨基酸，这些氨基酸与缺乏导致先天性角化不良的蛋白质保持一致。综上所述，来自这些不同技术的观察结果加在一起，超过了它们各部分的总和，结合在一起，对伪尿苷合成酶活性部位的事件有了更全面的了解，这些酶的生理重要性刚刚开始得到充分认识。

The pseudouridine synthases, found in all organisms, catalyze the isomerization of particular uridine residues in RNA to pseudouridine. Specific physiological roles for pseudouridine have also recently emerged: 1) pseudouridine is required in U2 snRNA for functional assembly of the U2 snRNP, which splices pre- mRNA; 2) the disease dyskeratosis congenita arises from the absence of a protein with sequence similarity to one pseudouridine synthase, TruB; 3) inhibition of pseudouridine synthases partly accounts for the cytotoxicity of 5-fluorouracil, an anti-cancer drug that is converted to 5-fluorouridine and incorporated into RNA in vivo. The mechanistic investigation of two pseudouridine synthases, TruB and RluA, is proposed. Events at the active site will be elucidated by integration of the information provided by site- directed mutagenesis, chemical modification, determination of kinetic isotope effects, and the direct observation of the adduct between pseudouridine synthases and RNA containing 5- fluorouridine using NMR methods. Site-directed mutagenesis revealed the catalytic importance of an aspartic acid, and site- specific chemical modification will probe the catalytic role played by that critical aspartic acid. Kinetic isotope effects will delineate the reaction step(s) affected by this amino acid. The chemical structure of the adduct between RNA containing 5- fluorouridine and the mutant protein will be determined and compared to that with the wild-type enzyme. This comparison will reveal the nature of protein-RNA interaction by identifying the particular groups in both the protein and the RNA that participate in the interaction, which is critical for understanding both pseudouridine synthase catalysis and the action of 5-fluorouracil. These studies will be expanded to examine more amino acid residues implicated in catalysis, including cysteine residues and the amino acids of TruB that align with the protein whose absence leads to dyskeratosis congenita. Taken together, the observations from these diverse techniques add up to more than the sum of their parts, combining to generate a fuller understanding of events at the active sites of pseudouridine synthases, enzymes whose physiological importance is just beginning to be fully appreciated.