Affinity Labeling of Nucleotide Sites in Proteins

核苷酸在细胞代谢中具有多种功能。例如，它们直接参与激酶参与激酶反应，它们作为辅酶分子的一部分参与脱氢酶反应，它们作为许多变紧酶的调节剂，它们参与（通过环AMP的作用）几种激素的作用，它们有助于血小板的聚集。本研究的目的是开发一种工具，该工具将允许系统地探索蛋白质核苷酸位点中的氨基酸残基。亲和标记的一般方法将基于本实验室先前合成的试剂，以及在嘌呤或核糖环烷基化剂的不同位置上能够与蛋白质共价反应的新试剂；因此，核苷酸片段将为结合位点内蛋白质的化学修饰提供特异性。这些核苷酸烷基化剂对腺苷琥珀酸合成酶的影响，以及对腺苷琥珀酸裂解酶的影响，将被检查。此外，还将探讨cAMP衍生物与血小板cAMP磷酸二酯酶的反应。血小板的聚集是由ADP诱导的，可能是通过与血小板表面膜上的受体位点结合而引起的。与嘌呤核苷酸类似物的亲和标记将用于表征血小板膜的ADP受体蛋白。对于每种蛋白质，将检查亲和标记反应的动力学，确定试剂掺入的程度，并从标记酶的蛋白水解酶中分离放射性肽以鉴定修饰残基。将对修饰酶的催化和配体结合性能以及构象特性进行评价。我们描述的第一类核苷酸类似物，氟磺酰基苯甲酰核苷，已经被用于在50多种蛋白质的特定位点发生反应；我们开发的新化合物很可能同样在标记蛋白质中的核苷酸位点方面具有广泛的适用性，并且这些研究将作为使用这些化合物探测其他酶的原型。此外，这些核苷酸类似物将在生物技术工业中有应用，它们可以用来检测克隆核苷酸结合蛋白的表达，并为合理设计药物提供知识基础，这些药物作为针对特定核苷酸需要酶的特定抑制剂。核苷酸在细胞代谢中具有多种功能。例如，核苷酸直接参与许多酶促反应，它们作为许多调节酶的激活剂或抑制剂，“第二信使”环AMP执行几种激素的作用。本研究的目的是开发一种工具，该工具将允许系统地探索结合核苷酸的酶的区域。“亲和标记”的一般方法将被使用，在这种方法中，我们将能够与天然核苷酸形成永久连接的反应部分（称为烷基化剂）附加到天然核苷酸上。新试剂的核苷酸部分将化合物导向蛋白质上的特定结合位点，反应性部分将使结合不可逆，从而可以分离和表征结合位点。我们现在将研究这些核苷酸烷基化剂对嘌呤核苷酸生物合成的两种关键酶和对调节核苷酸环AMP降解的一种重要酶的影响。我们描述的第一类核苷酸类似物，氟磺酰基苯甲酰核苷，已经被用于在50多种蛋白质的特定位点发生反应；我们开发的新化合物很可能在实验室中对蛋白质中的核苷酸位点具有类似的适用性，这些研究将作为使用这些化合物探测其他酶的原型。此外，这些核苷酸类似物将在生物技术工业中有应用，它们可以用来检测克隆核苷酸结合蛋白的表达，并为合理设计药物提供知识基础，这些药物作为针对特定核苷酸需要酶的特定抑制剂。＊＊＊

9423108 Colman Nucleotides have a multi-functional role in cellular metabolism. For example, they are directly involved in kinase involved in kinase reactions, they participate in dehydrogenase reactions as part of the coenzyme molecule, they function as regulators of many allosteric enzymes, they are involved (through the effects of cyclic AMP) in the action of several hormones, and they contribute to the aggregation of platelets. The aim of this study is to develop the tools which will allow a systematic exploration of the amino acid residues in the nucleotide sites of proteins. The general approach of affinity labeling will be used based on reagents previously synthesized in this laboratory as well as on new reagents which incorporate, at various positions of the purine or ribose ring alkylating agents capable of reacting covalently with the protein; the nucleotide moiety will thus provide the specificity for chemical modification of the proteins within the binding sites. The effects of these nucleotide alkylating agents on adenylosuccinate synthetase, as well as on adenylosuccinate lyase, will be examined. Further, the reactions of cAMP derivatives with platelet cAMP phosphodiesterases will be explored. The aggregation of blood platelets is induced by ADP, presumably by binding to receptor sites on the platelet surface membrane. Affinity labeling with the purine nucleotide analogues will be used to characterize the ADP receptor protein of platelet membranes. For each protein, the kinetics of the affinity labeling reaction will be examined, the extent of reagent incorporation will be determined and radioactive peptides will be isolated from proteolytic digests of labeled enzyme to identify the modified residue. The catalytic and ligand binding properties as well as the conformational characteristics of the modified enzyme will be evaluated. The first class of nucleotide analogues which we described, the fluorosulfonylbenzoyl nucleosides, has already been used to react at specific sites of more than 50 proteins; it is likely that the newer compounds which we develop will similarly have broad applicability in the labeling of nucleotide sites in proteins and that these studies will serve as prototypes for the use of these compounds to probe other enzymes. In addition, these nucleotide analogues will have applications in the biotechnology industry where they can be used to detect expression of cloned nucleotide binding proteins, and to provide the knowledge base for rational design of drugs functioning as specific inhibitors targeted to particular nucleotide-requiring enzymes. %%% Nucleotides have a multifunctional role in cellular metabolism. For example, nucleotides participate directly in numerous enzymatic reactions, they function as activatos or inhibitors of many regulatory enzymes, and the"second messenger" cyclic AMP carries out the action of several hormones. The aim of this study is to develop the tools which will allow a systematic exploration of the region of an enzyme which binds nucleotides. The general approach of "affinity labeling" will be used in which we attach to a natural nucleotide a reactive portion (called an alkylating agent) which is capable of forming a permanent link to a protein. The nucleotide moiety of the new reagent will direct the compound to a specific binding site on the protein, and the reactive portion will make the binding irreversible so that the binding site can be isolated and characterized. We will now study the effects of these nucleotide alkylating agents ontwo enzymes ctitical for purine nucleotide biiosynthesis and on an enzyme important in the degradation of the regulatory nucleotide cyclic AMP. The first class of nucleotide analogues which we described, the fluorosulfonylbenzoyl nucleosides, has aleady been used to react at specific sites of more than 50 proteins; it is likely that the newer compounds which we develop will similarly have b road applicability in the lab eling of nucleotide sites in proteins and that these studies will serve as prototypes for the use of these compounds to probe other enzymes. In addition, these nucleotide analogues will hve applications in the biotechnology industry where they can be used to detect expression of cloned nucleotide binding proteins, and to provide the knowledge base for rational design of drugs functioning as specific inhibitors targeted to particular nucleotide-requiring enzymes. ***