ENZYME ACTIVE SITE MAPPING UTILIZING CARBONIUM IONS

我们的长期目标是绘制酶的活性部位图 活性位导向的酶激活抑制剂，可以提供 高活性的碳正离子到活性部位。的完整地图 一种酶可以让人准确地定位细胞的释放点 碳离子；变量的系统变化将允许一个人 了解不同的抑制剂将如何具有不同的释放点 (反映了它们不同的绑定)以及这些释放点 会随pH值等而改变。关于凝乳酶， 使用的程序是：抑制、还原和烷基化，G-75 葡聚糖多肽的分离、测序 作为酰胺键O-烷基化的结果而形成的碎片， 对完整的C链进行化学修饰以增加溶解度， 胰酶和胰凝乳酶消化，多肽的高效液相分离， 完全水解，氨基酸分析，合成可疑的N- 苯基氨基酸，并用层析法比较 识别后者的标准和未知数。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置 关于氧(丝氨酸214的羰基)，并做了一个实践 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--作为这方面的一项主要任务 提案-将通过我们的方法完成第一个测绘案例。 已经提出了一个假说来解释我们人类的活动 D-家族抑制剂对凝乳酶的抑制作用。我们计划 在设计、合成和测试中验证该假设 胰酶抑制剂。我们将使用13C核磁共振波谱来 指导我们在蛋白质方面的工作，以鉴定某些苄基 取代氨基酸，并进行后续化学反应 使用修饰过的酶。我们的方法导致了卵裂 多肽链；亚硝酸内酰胺和亚硝磺胺抑制剂将 为了控制卵裂的部位(S)进行了检查。最后， 将尝试使用荧光标签。这个 我们使用的抑制剂与抗癌药物密切相关 更重要的是，已知抗蛋白酶有潜在的用途 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.