MECHANISTIC ENZYMOLOGY OF HIV PROTEINS

酶学、化学和酶动力学方法，以及合成， 物理化学和分析化学正在被用来研究 青枯菌逆转录酶和蛋白酶的作用机制 HIV-1，最终目标是开发针对这些病毒的特定抑制剂 酵素。反转录病毒蛋白水解酶和P‘s的光学测定法 这个实验室正被用来研究反应的影响 逆转录酶和逆转录酶活性的条件和潜在的抑制剂 逆转录病毒蛋白酶。I)连续紫外分光光度测定法 在以前的报告中描述的是用来研究伸长的，催化的 由大肠杆菌DNA聚合酶I的Klenow片段部分 由35个碱基组成的自互补“发夹”型寡核苷酸 单核苷酸残基。该方法的敏感性允许 单一碱基结合的量度。二)浓度 高达4.5M的氯化钠加速了一种显色剂的水解 禽成髓细胞增多症病毒蛋白酶催化的多肽底物。 我们对一种多肽的裂解也观察到了类似的效果 由HIV-1蛋白酶催化的底物，也有报道由 另一些用于这种酶对几种多肽的裂解。这些盐效应 似乎与亚基的关联无关，而亚基是 逆转录病毒天冬氨酸蛋白酶的活性，因为行为非常相似 是用哺乳动物的胃酶单体观察到的。三)我们有 在RT中发现了两个氨基酸重复基序，这两个基序在10 HIV-1和8个HIV-2分离株，它们出现在蛋白质的一个区域 可能与p66和p51亚基的关联有关 酵素。一种图案是经过修改的“亮氨酸拉链”，而另一种图案是 涉及五种色氨酸。由于HIV-1RT的活性一直是 建议要求亚基关联，了解这一过程 可能导致抑制这种酶的新方法。

Methods of enzymology, chemical and enzymatic kinetics, and synthetic, physical and analytical chemistry are being used to investigate the mechanisms of action of reverse transcriptase (RT) and protease enzymes of HIV-1, with the ultimate goal of developing specific inhibitors for these enzymes. Optical assays for retroviral proteases and P's, developed in this laboratory, are being employed in studies of the effects of reaction conditions and potential inhibitors on the activity of both RT and retroviral proteases. i) A continuous UV spectrophotometric assay described in a previous Report was used to study the elongation, catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I, of a partially self-complementary "hairpin" shaped oligonucleotide consisting of 35 mononucleotide residues. The sensitivity of the method permits the measurement of incorporation of a single base. ii) Concentrations of sodium chloride up to 4.5 M accelerated the hydrolysis of a chromogenic peptide substrate catalyzed by the protease of avian myeloblastosis virus. Similar effects have been observed by us for the cleavage of a peptide substrate catalyzed by HIV-1 protease, and have also been reported by others for cleavage of several peptides by this enzyme. These salt effects appear to be unrelated to the association of subunits that is required for the activity of retroviral aspartyl proteases, since very similar behavior is observed with the monomeric, mammalian enzyme pepsin. iii) We have identified two amino-acid repeat motifs in RT which are conserved in ten HIV-1 and eight HIV-2 isolates, and which occur in a region of the protein that may be involved in association of the p66 and p51 subunits of this enzyme. One motif is a modified "leucine zipper", whereas the other motif involves five tryptophans. Since the activity of HIV-1 RT has been suggested to require subunit association, an understanding of this process may lead to novel approaches to the inhibition of this enzyme.