MECHANISMS OF PEPSIN CATALYSIS & INHIBITION

该项目将提供有关以下方面的新信息： 底物和酶在酶活性位点的相互作用 天冬氨酸蛋白酶家族。 我们认为， 胃蛋白酶样酶的kcat通过氢来控制 与底物的肽基团的主链键合， Km的水平由与侧链基团的相互作用决定 的基板。 我们获得的新信息将是有价值的 在设计与控制消化性溃疡有关新药时 （胃蛋白酶），高血压（肾肾素），和预防 HTLV-III的病毒成熟， 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充分利用 新的实验结果，从我们的动力学研究以及x射线 晶体学数据和克隆和测序数据来自其他 laboratories. 在上一个资助期， 利用确定的合成发色底物的动力学测定。 根据这些底物水解的pH依赖性， 新的互动被揭示出来。 的可用性 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， 使酶残留物发生明确的变化， 重要联系人 已经安排了与几个 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。 结合1）简便的测定，2）灵敏的测试， 通过研究动力学的pH依赖性，3） 大大提高了合成底物变体的能力， 结构，4）获得选择性改变的互补能力 在酶残基中，5）高分辨率晶体结构， 和6）关于潜在相互作用的新一代想法， 活性位点分裂应该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.