ENZYME ACTIVE SITE MAPPING UTILIZING CARBONIUM IONS

我们的长期目标是利用 活性位点定向的酶激活抑制剂， 高活性碳正离子的活性位点。 完整的地图为 一种酶可以让人们精确地定位 碳正离子;变量的系统变化将允许人们 看看不同的抑制剂有不同的释放点 （反映了它们不同的约束力）以及这些释放点 将随pH值等而变化。关于胰凝乳蛋白酶， 使用的程序是：抑制，还原和烷基化，G-75 肽的葡聚糖凝胶分离，肽的测序 由于酰胺键的O-烷基化而形成的片段， 化学修饰完整的C链以增加溶解度， 胰蛋白酶和胰凝乳蛋白酶消化，肽的HPLC分离， 充分水解，氨基酸分析，合成疑似N- 苄基氨基酸，和色谱比较， 标准和未知数来识别后者。 对于“映射” 对于α-糜蛋白酶，我们已经定位了烷基化的主要位点 氧（丝氨酸214的羰基），并已作出了实践 运行以确定稳定标签的位置（在N、S和C上）; 完成后一个方面-作为一个主要的推力， 建议-将完成我们的方法映射的第一个案例。 一个假设已经发展到解释我们的活动， D-家族抑制剂对胰凝乳蛋白酶的抑制作用。 我们计划 在设计、综合和检验中检验假设， 胰蛋白酶抑制剂。 我们将使用13 C NMR光谱， 指导我们在蛋白质工作中， 取代的氨基酸，并遵循化学反应进行 在改良酶上。 我们的方法导致分裂的 肽链;亚硝基内酰胺和亚硝基磺内酰胺抑制剂将被 检查以控制切割位点。 最后， 将尝试利用荧光标记。 的 我们使用的抑制剂与抗癌药物密切相关， 活性;此外，已知抗蛋白酶具有潜在的用途 在医学上。

Our long term objective is to map the active sites of enzymes using activesite-directed enzyme-activated-inhibitors which can deliver highly active carbonium ions to the active sites. The full map for an enzyme will allow one to pinpoint the release point of the carbonium ion; systematic changes of variables will allow one to see how different inhibitors will have different release points (reflecting their different binding) and how those release points will change with pH, etc. With reference to chymotrypsin, the procedures used are: inhibition, reduction and alkylation, G-75 Sephadex separation of the peptides, sequencing of the peptide fragments formed as a result of O-alkylation of amide linkages, chemical modification of intact C-chain to increase solubility, tryptic and chymotryptic digestion, HPLC separation of peptides, full hydrolysis, amino acid analysis, synthesis of suspected N- benzyl amino acids, and chromatographic comparisons of the standards and unknowns to identify the latter. For the "mapping" of alpha-chymotrypsin, we have located the major site of alkylation on oxygen (carbonyl group of serine 214) and have made a practice run to determine the location of the stable labels (on N,S, and C); completion of the latter aspect - as a major thrust of this proposal - will complete the first case of mapping by our approach. A hypothesis had been developed to account for the activity of our D-family inhibitors in the inhibition of chymotrypsin. We plan to test that hypothesis in design and synthesis and testing of inhibitors for Trypsin. We will be using 13C NMR spectroscopy to guide us in the protein work, to identify certain benzyl substituted amino acids, and to follow chemical reactions carried out on the modified enzyme. Our method leads to the cleavage of peptide chains; nitrosolactam and nitrososultam inhibitors will be examined in an effort to control the site(s) of cleavage. Finally, attempts will be made to utilize fluorescent labels. The inhibitors we use are closely related to those with anti-cancer activity; further; anti-proteases are known to have potential uses in medicine.