STRUCTURE-FUNCTION STUDIES OF THE HIV PROTEASE

HIV结构与功能关系的基础研究 蛋白酶（艾滋病毒公关）将进行的目标是增加我们的 了解这种重要的治疗酶。 高度优化 全化学合成将用于制备HIV PR以及 一系列合成的酶类似物 这些类似物将 合理设计，使用分子建模来探测重要的 结构-功能关系隐含在酶的拟议模式 的行动。 在整个计划中， 这些修改的热力学影响将被衡量在一些 的方式。 适当同位素的完整2D-NMR分配 标记的合成酶将进行，并随后用作 结构指纹，通过它来常规地比较类似物。 此外，预计热力学和动力学 将对所有合成酶进行测量，同样，这些 量将与天然酶进行比较。 此外，X射线 将对选定的类似物进行晶体学分析。 动态 核磁共振实验的性质非常适合皮瓣的研究 在底物结合时明显发生的运动。 拟议 水301在催化机制中的作用将通过现场评估- 被认为参与氢键的肽键的特异性置换 通过这种水分子与底物/抑制剂相互作用。 的 催化活性Asp 25、Asp 125侧链的电离状态 将通过用13 C作为NMR的单原子标记直接测定 记者组。 将推导出控制底物特异性的规则， 并将通过构建类似的PR分子进行测试， 填充和/或电荷改性几何约束部分 引入P1和P1'特异性口袋。 根本 从这些HIV PR研究中获得的知识对于以下方面很重要： 对相关临床相关酶的理解，如 其它逆转录病毒蛋白酶和细胞编码的N-乙酰基蛋白酶， 作为肾素。

Fundamental studies of structure-function relationships of the HIV Protease (HIV PR) will be undertaken with the goal of increasing our understanding of this therapeutically important enzyme. Highly optimized total chemical synthesis will be used to prepare both HIV PR as well as a series of synthetic analogues of the enzyme. These analogues will be rationally designed using molecular modeling to probe the important structure-function relationships implicit in the enzyme's proposed mode of action. Throughout this program the fundamental structural and thermodynamic impact of these modifications will be gauged in a number of ways. The complete 2D-NMR assignment of an appropriately isotopically labeled synthetic enzyme will be undertaken and subsequently used as a structural fingerprint by which to routinely compare analogues. Furthermore, it is anticipated that thermodynamic and kinetic measurements will be made on all synthetic enzymes, again these quantities will be compared with the native enzyme. In addition, X-ray crystallography will be performed on selected analogues. The dynamic nature of the NMR experiment is ideally suited to the study of the flap movements which apparently occur upon substrate binding. The proposed role of Water301 in the catalytic mechanism will be evaluated by site- specific replacement of peptide bonds thought to be involved in H-bonding interactions with substrates/inhibitors through this water molecule. The ionization state of the catalytically active Asp25, Asp125 side chains will be directly determined by single-atom labeling with 13C as an NMR reporter group. Rules governing substrate specificity will be deduced, and will be tested by construction of analog PR molecules with space filling and/or charge modifying geometrically-constrained moieties introduced into the P1 and P1' specificity pockets. The fundamental knowledge resulting from these studies of HIV PR will be important for the understanding of related clinically-relevant enzymes, such as the other retroviral proteases, and of cell-encoded aspartyl proteinases such as renin.