STRUCTURE-FUNCTION STUDIES OF THE HIV PROTEASE

HIV结构与功能关系的基础研究 将进行蛋白水解酶(HIV PR)，目标是增加我们的 了解这种具有重要治疗作用的酶。高度优化 全化学合成将用于制备HIV PR以及 一系列酶的合成类似物。这些类似物将是 合理设计利用分子模拟来探索重要 酶的建议模式中隐含的结构-功能关系 行动的一部分。在整个计划中，基本的结构和 这些修改的热力学影响将以数字形式进行测量 当然了。适当同位素的全2D-核磁共振指认 标记的合成酶将被用于随后作为 用于常规比较类似物的结构指纹。 此外，预计热力学和动力学 将对所有合成酶进行测量，同样是这些 数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结晶学研究。动态感 核磁共振实验的性质非常适合研究襟翼 显然发生在底物结合时的运动。建议数 水301在催化机理中的作用将通过现场- 被认为与氢键有关的多肽键的特定取代 通过这种水分子与底物/抑制剂相互作用。这个 催化活性Asp25、Asp125侧链的电离态 将用~(13)C作为核磁共振单原子标记直接测定 记者团。将推导出控制底物专一性的规则， 并将通过构建具有空间的模拟PR分子进行测试 填充和/或电荷修饰几何约束部分 被引入到P1和P1的专一性口袋中。最基本的 从这些艾滋病毒公关研究中获得的知识对 对相关临床相关酶的了解，如 其他逆转录病毒蛋白酶，以及细胞编码的天冬氨酸蛋白酶，如 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.