MECHANISMS OF PEPSIN CATALYSIS & INHIBITION

该项目将提供有关以下细节的新信息 底物与酶活性部位的相互作用 天冬氨酸蛋白水解酶家族。我们认为这一水平 KCAT对胃蛋白酶样酶的作用是通过氢控制的 结合到底物的多肽基团的骨架上，同时 Km的水平主要由与侧链基团的相互作用所决定 底物的一部分。我们获得的新信息将是有价值的 与消化性溃疡控制相关的新药设计 (胃蛋白酶)、高血压(肾肾素)和预防 HTLV-III病毒成熟，疑似 获得性免疫缺陷综合征(逆转录病毒)的病理生理学 蛋白酶)。 我们实现这些目标的方法将利用非常 来自我们的动力学研究和x射线的新实验结果 结晶学数据和克隆和测序数据 实验室。在之前的授权期内，我们改进了我们的 利用已定义的合成发色底物的动力学分析。 从这些底物的pH依赖关系来看，有几种 新的相互作用被揭示出来。随着可用 定点突变，现在可以测试我们的 关于这些相互作用的重要性的假设通过 对怀疑有贡献的酶残留物进行明确的改变 重要的联系人。已经与几家公司安排了合作 在克隆必要基因方面取得进展的实验室 或确定关键酶的高分辨率X射线结构。 1)简便试验，2)敏感性试验相结合 通过研究动力学的pH依赖性来研究相互作用，3) 极大地提高了合成底物变体的能力 结构，4)互补能力，以获得选定的更改 在酶残基中，5)高分辨晶体结构， 和6)关于潜在相互作用的新一代想法 积极的场地裂隙应该会使这个项目非常成功。

This project will provide new information on details of interactions between substrate and enzyme at the active site of the Aspartic Proteinase family of enzymes. We believe that the level of kcat for pepsin-like enzymes is controlled through hydrogen bonding to the backbone of peptide groups of the substrates while the level of Km is dominated by interactions with side-chain groups of the substrate. The new information we obtain will be valuable in design of new drugs relevant to control of peptic ulcers (gastric pepsin), hypertension (kidney renin), and prevention of viral maturation of HTLV-III, the virus suspected in the pathophysiology of Acquired Immune Deficiency Syndrome (retroviral protease). Our approach to achieving these ends will take advantage of very new experimental results from our kinetic studies as well as x-ray crystallographic data and cloning and sequence data from other laboratories. ln the previous grant period we have refined our kinetic assay utilizing defined synthetic, chromophoric substrates. From the pH dependence of hydrolysis of these substrates, several new interactions have been revealed. With the availability of site-directed mutagenesis, it is now possible to test our hypotheses about the importance of these interactions in detail by making defined changes in enzyme residues suspected of contributing vital contacts. Collaborations have been arranged with several laboratories that have made progress in cloning the necessary genes or determining the high resolution x-ray structure of key enzymes. The combination of a 1) facile assay, 2) sensitive test for interactions by a study of the pH dependence of kinetics, 3) greatly improved ability to synthesize variants of the substrate structure, 4) complementary ability to obtain selected alterations in enzyme residues, 5) high resolution crystallographic structures, and 6) new generation of ideas about potential interactions in the active site cleft should make this project highly successful.