MECHANISTIC ENZYMOLOGY OF HIV PROTEINS

酶学，动力学和合成，物理和分析化学是 用于研究催化和组装的机制 人类免疫缺陷病毒逆转录酶和蛋白酶 1型病毒（HIV-1），最终目标是开发特异性 这些酶的抑制剂。 （一）在上一份报告中，我们描述了一个 氨基酸重复基序，类似于修饰的"亮氨酸拉链"，在RT 几个HIV-1和HIV-2分离株，我们认为可能涉及 在亚基联合中。 Kohlstaedt等人的X射线研究现在已经 表明p51和p66中含有该基序的区域 亚基在结晶异二聚体中不接触。 因此 这种高度保守序列的功能尚未确定。 ii）第二阶段 氯化钠加速多肽水解速率的研究 由逆转录病毒蛋白酶以及模型催化的水解 哺乳动物的酶胃蛋白酶已经完成 这种盐效应， 几乎完全取决于米氏常数，建议由 基质和膜之间疏水相互作用的增强 酶的活性部位。 使用分光光度测定法， 评估底物的溶解度，我们已经观察到一个单调增加 NaCl浓度高达5 M。 有文献报道说- 速率对盐浓度的形状依赖性可能是由以下原因引起的： 由于未能检测到底物溶解度降低而产生的伪影 在高盐浓度下。 iii）自动加工的动力学研究 含有侧翼Pol区的HIV-1蛋白酶的构建体 序列，并表达为与麦芽糖结合的融合蛋白。 大肠杆菌蛋白质的研究进展。 初步结果表明 含有麦芽糖结合的N-末端序列的初始丢失 蛋白质，得到13.2-kDa的中间体，其保留C-末端Pol 该中间体在较慢的酶切条件下被切割成11-kDa蛋白酶。 步 在所用浓度下，初始裂解是一级裂解， 蛋白质浓度，与快速和有利的二聚化一致 随后进行分子内蛋白水解。

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.