MECHANISTIC ENZYMOLOGY OF HIV PROTEINS

酶学、动力学、合成、物理和分析化学 用来研究其催化和组装的机理 人类免疫缺陷的逆转录酶和蛋白酶 1型病毒(HIV-1)，最终目标是开发出特定的 这些酶的抑制剂。I)在之前的一份报告中，我们描述了 RT中的氨基酸重复基序，类似于修改的“亮氨酸拉链” 几个HIV-1和HIV-2分离株，我们认为这可能与 在亚基关联中。Kohlstaedt等人的X射线研究。现在有了 表明在p51和p66中包含该基序的区域 结晶杂二聚体中的亚基不接触。因此， 这个高度保守的序列的功能尚未确定。Ii) 氯化钠对多肽生成速率的加速作用研究 逆转录病毒蛋白水解酶催化的水解酶及其模型 哺乳动物的酶，胃酶，现在是完整的。这种盐效应，也就是 几乎完全依赖于Michaelis常数，建议由 增强了底物和底物之间的疏水相互作用 酶的活性部位。使用分光光度分析，这有助于 评估底物的溶解度，我们观察到单调的增加 在氯化钠浓度高达5M的情况下，文献报道了钟声- 这种速率与盐浓度的形状依赖关系可能是由于 由于未能检测到底物的溶解度降低而产生的伪影 在高盐浓度下。3)自动加工的动力学研究 含有侧翼Pol区的HIV-1蛋白酶的构建 测序并以麦芽糖结合融合蛋白的形式表达 大肠埃希氏菌的蛋白质正在研究中。初步结果表明 含有麦芽糖结合的N-末端序列的初始损失 提供13.2 kDa中间体的蛋白质，该中间体保留C-末端的极点 该中间体以较慢的速度被切割到11-kDa的蛋白水解酶 一步。在所使用的浓度下，初始切割是一级的 蛋白质浓度，与快速和有利的二聚化一致 对融合蛋白进行分子内蛋白降解。

Enzymology, kinetics and synthetic, physical and analytical chemistry are being used to investigate the mechanisms of catalysis by and assembly of the reverse transcriptase (RT) and protease of human immunodeficiency virus type 1 (HIV-1), with the ultimate goal of developing specific inhibitors for these enzymes. i) In a previous report, we described an amino-acid repeat motif, resembling a modified "leucine zipper", in the RT of several HIV-1 and HIV-2 isolates, which we suggested might be involved in subunit association. X-ray studies by Kohlstaedt et al. have now indicated that the regions containing this motif in the p51 and p66 subunits are not in contact in the crystalline heterodimer. Thus, the function of this highly conserved sequence is as yet undefined. ii) Studies on the acceleration by sodium chloride of the rate of peptide hy- drolysis catalyzed by retroviral proteases as well as by the model mammalian enzyme, pepsin, are now complete. This salt effect, which is almost exclusively on the Michaelis constant, is suggested to result from the enhancement of hydrophobic interactions between the substrate and the enzyme's active site. Using a spectrophotometric assay, which facilitated assessment of substrate solubility, we have observed a monotonic increase in rate with NaCl concentrations up to 5 M. Literature reports of a bell- shaped dependence of the rate on salt concentration presumably result from artifacts due to the failure to detect reduced solubility of the substrate at high salt concentrations. iii) Kinetic studies on the autoprocessing of constructs of the HIV-1 protease containing flanking Pol region sequences and expressed as fusion proteins with the maltose-binding protein of Escherichia coli are in progress. Preliminary results suggest an initial loss of the N-terminal sequence containing the maltose-binding protein to give a 13.2-kDa intermediate which retains the C-terminal Pol sequence; this intermediate is cleaved to the 11-kDa protease in a slower step. At the concentrations used, the initial cleavage is first-order in protein concentration, consistent with a rapid and favorable dimerization of the fusion protein followed by intramolecular proteolysis.