Affinity Labeling of Nucleotide Sites in Proteins

科尔曼9728202核苷酸在细胞代谢中具有多种功能：参与激酶反应，作为辅酶分子的一部分参与脱氢酶反应，作为许多变质酶的调节剂，（通过环AMP的作用）参与几种激素的作用，并有助于血小板的聚集。本研究的目的是开发工具，以系统地探索氨基酸残基的核苷酸位点的蛋白质。使用亲和的一般方法，使用本实验室先前合成的试剂和在嘌呤或核糖环的不同位置加入能够与蛋白质共价反应的烷基化剂的新试剂；因此，核苷酸片段为结合位点内蛋白质的化学修饰提供了特异性。研究了这些核苷酸烷基化剂对几种酶的影响；腺苷琥珀酸合成酶和腺苷琥珀酸裂解酶，催化一磷酸肌苷转化为AMP的连续反应；以及环AMP磷酸二酯酶，它催化cAMP水解形成5' - AMP。对于每种蛋白质，研究了亲和标记反应的动力学，确定了试剂掺入的程度，并从标记酶的蛋白水解酶中分离出放射性肽，并与修饰的残留物进行了鉴定。并对改性酶的催化和配体结合性能以及构象特性进行了评价。对于大多数这些酶，这些酶在溶液中的结果与它们确定的晶体结构进行了比较。核苷酸类似物氟磺酰基苯甲酰核苷已经在50多种蛋白质上进行了测试；结果表明，该方法对其他酶具有普遍的适用性。它们可用于克隆表达的核苷酸结合蛋白的检测。这项研究的意义可能在于为合理设计针对特定核苷酸需要酶的特定抑制剂提供了k知识库。2. 非技术核苷酸在细胞代谢中具有多种功能，如参与酶促反应，激活调节酶，并作为“第二信使”，如环AMP。本研究旨在开发工具，允许系统地探索结合核苷酸的酶。一般采用“亲和标记”的方法，将新试剂化合物的核苷酸片段不可逆地附着在蛋白质上的特定结合位点上，从而分离和表征该结合位点。研究了核苷酸烷基化剂对两种嘌呤核苷酸生物合成酶和一种降解核苷酸环AMP的重要酶的影响。核苷酸类似物，如氟磺酰基苯甲酰核苷，在特定位点与蛋白质反应。对于生物技术，这些分子可以用来检测克隆蛋白的表达，并为合理设计核苷酸需要酶的抑制剂提供知识基础。

Colman 9728202 1. Technical Nucleotides have a multi-functional role in cellular metabolism: involved in kinase reactions, participate in dehydrogenase reactions as part of the coenzyme molecule, function as regulators of many allosteric enzymes, engaged (through the effects of cyclic AMP) in the action of several hormones, and contribute to the aggregation of platelets. This study is aimed to develop tools for a systematic exploration of the amino acid residues in the nucleotide sites of proteins. The general approach of affinity is used, with reagents previously synthesized in this laboratory and 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 thus provides the specificity for chemical modification of the proteins within the binding sites. The effects of these nucleotide alkylating agents are examined on several enzymes; adenylosuccinate synthetase and adenylosuccinate lyase, which catalyze successive reactions in the conversion of inosine monophosphate to AMP; and cyclic AMP phosphodiesterases, which catalyzes the hydrolysis of cAMP to form 5' - AMP. For each protein, the kinetics of the affinity labeling reaction is examined, the extent of reagent incorporation determined and the radioactive peptides isolated from proteolytic digests of labeled enzyme and identified with the modified residue. The catalytic and ligand binding properties as well as the conformational characteristics of the modified enzymes are also evaluated. For most of these enzymes, the results of these enzymes in solution are compared with their determined crystal structures. The nucleotide analogs, fluorosulfonylbenzoyl nucleosides, have been tested on more than 50 proteins; with results promising for its general applicability to other enzymes. They are useful for the detection of nucleotide binding proteins expressed by cloning. The significance of this may rest with providing the k nowledge base for rational design of specific inhibitors targeted to particular nucleotide-requiring enzymes. 2. Non-technical Nucleotides have a multifunctional role in cellular metabolism, such as participating in enzymatic reactions, activating regulatory enzymes, and serving as "second messenger" like cyclic AMP. This study is aimed to develop the tools which allow a systematic exploration of the enzyme which binds nucleotides. A general approach of "affinity labeling" is used to attach a nucleotide moiety of the new reagent compound to a specific binding site on the protein, irreversibly, so that the binding site can be isolated and characterized. The effects of nucleotide alkylating agents on two purine nucleotide biosynthesis enzymes and on an enzyme important in the degradation of nucleotide cyclic AMP are studied. Nucleotide analogues such as fluorsulfonylbenzoyl nucleosides which react to proteins at specific sites are specifically probed. For biotechnology, these molecules can be used to detect expression of cloned proteins, and to provide the knowledge base for rational design of inhibitors for nucleotide-requiring enzymes.