HUMAN IMMUNODEFICIENCY VIRUS PROTEINASE

该项目将提供有关催化剂的新信息， 人免疫缺陷病毒蛋白水解酶的性质 病毒，HIV-PR。这是一个重要的催化活性， 感染性病毒颗粒的复制和成熟， 作为抗病毒治疗靶点。 我们将制备一系列寡肽底物， 验证了底物结构和活性中心的几种理论 特性. 两个主要的新建议是，基板必须 以a）在切割点的β转角构象存在， 或B）双链β折叠二聚体， 酶-底物相互作用发生。 这些理论是由 根据观察，虽然简单的线性寡肽 被分离的酶切割，观察到的动力学参数 最多也就是贫血 我们将设计限制性肽， Cys残基在特定的点，以有利于形成顺式 或环状的反式Tyr-Pro肽键，二硫键连接 肽。 为了测试二聚体形成的替代理论，我们将 构建平行以及反平行二聚体的策略 Cys和Lys残基的位置。 在这两种情况下， 一种类似于病毒中天然结构的构象 多聚蛋白应该导致大大增强的结合或催化 特性. 最后，我们将根据这些新信息， 包括发色基团，以产生连续 分光光度法测定这一重要酶。 该测定 对于表征潜在的抗- 病毒抑制剂

This project will provide new information on the catalytic properties of the proteolytic enzyme of the Human Immunodifficiency Virus, HIV-PR. This is an essential catalytic activity for the replication and maturation of infectious virus particles and is a target for antiviral therapy. We will prepare a series of oligopeptide substrates designed to test several theories of substrate structure and active site properties. The two main new proposals are that the substrate must exist in either a) a beta turn conformation at the cleavage point, or b) a two-stranded beta sheet dimer in order for productive enzyme-substrate interaction to occur. These theories are derived from the observations that, although simple linear oligopeptides are cleaved by the isolated enzyme, the observed kinetic parameters are anemic at best. We will design constrained peptides by placing Cys residues at specific points to favor formation of either a cis or trans Tyr-Pro peptide bond in a cyclic, disulfide linked peptide. To test the alternate theory of dimer formation, we will construct parallel as Well as anti-parallel dimers by strategic location of Cys and Lys residues. In both cases, construction of a conformation that resembles the native structure in the viral polyprotein should lead to greatly enhanced binding or catalytic properties. Finally, we will build upon this new information to include chromophoric groups in an effort to produce a continuous spectrophotometric assay for this important enzyme. This assay would be extremely valuable for characterization of potential anti- viral inhibitors.