PSEUDOURIDINE SYNTHASES

假尿嘧啶合成酶存在于所有生物体中，催化RNA中特定尿嘧啶残基异构化为假尿嘧啶。假尿嘧啶的特定生理作用最近也被发现：1)假尿嘧啶在U2 snRNA中是U2 snRNP拼接前mRNA的功能组装所必需的；2)先天性角化不良症是由于缺乏与假尿嘧啶合成酶TruB序列相似的蛋白质引起的；3) 5-氟尿嘧啶（5-fluorouracil）是一种在体内转化为5-氟吡啶并掺入RNA的抗癌药物，对假尿嘧啶合成酶的抑制是其细胞毒性的部分原因。提出了两种假尿嘧啶合成酶TruB和RluA的机理研究。在活性位点上发生的事件将通过整合位点定向诱变、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尿嘧啶合酶与含有5-氟尿嘧啶的RNA之间的加合物所提供的信息来阐明。位点定向诱变揭示了天冬氨酸的催化作用，位点特异性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述受该氨基酸影响的反应步骤。将测定含有5-氟吡啶的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参与相互作用的蛋白质和RNA中的特定基团来揭示蛋白质-RNA相互作用的本质，这对于理解假尿嘧啶合成酶的催化作用和5-氟尿嘧啶的作用至关重要。这些研究将扩展到更多与催化有关的氨基酸残基，包括半胱氨酸残基和TruB氨基酸，这些氨基酸与蛋白质一致，其缺失导致先天性角化不良。综合起来，这些不同技术的观察结果加起来比它们各自部分的总和还要多，结合起来，对假尿嘧啶合成酶活性位点的事件有了更全面的了解，这些酶的生理学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.