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。为了促进这些研究，逆转录病毒的光学分析 蛋白水解酶和逆转录酶已被开发出来。I)裂解生色物质 衬底、Ac-Lys-Ala-Ser-Gln-Asn-(p-nitro)PhePro-Val-Val-amide和 Thr-Phe-Gln-Ala-(p-nitro)Phe-Pro-Leu-Arg-Glu-Ala，的催化作用 HIV-1和禽成髓细胞增生症病毒(AMV)的逆转录病毒蛋白酶， 分别发生在对硝基苯丙氨基和对硝基苯丙氨基之间。 脯氨基残基。这些水解物伴随着紫外线的增加。 在316 nm处的吸收，允许方便、连续地监测 这些反应的进展。二)RT和其他DNA的光学分析 聚合酶已经被开发出来。这些化验是基于 圆二色谱(CD)和伸长时的紫外光谱 合成模板/底物双链的螺旋部分。在……面前 大肠杆菌DNA聚合酶I或HIV-1RT的Klenow片段，掺入 将DTMP合成由40-60-聚体DA组成的合成模板/底物， 用20聚体的DT预涂，在负CD带产生增强 在248 nm处，紫外光吸收在260-275 nm处有所下降。这两个变化 与DTMP的掺入程度呈线性关系。相似的结果 用寡核苷酸(DT)引发的聚(Ra)用HIV-1RT获得。这些 逆转录病毒酶的连续测定方法提供了一个明显的优势 在单点分析上，因为它们允许测量整个 单量60-120微升酶促反应的时间进程 样本。这些方法目前正被用于研究 反应条件和特定官能团试剂对反应的影响 RT和HIV-1蛋白水解酶的活性。

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. To facilitate these studies, optical assays for retroviral proteases and RTs have been developed. i) Cleavage of the chromogenic substrates, Ac-Lys-Ala-Ser-Gln-Asn-(p-nitro)PhePro-Val-Val-amide and Thr-Phe-Gln-Ala-(p-nitro)Phe-Pro-Leu-Arg-Glu-Ala, catalyzed by the retroviral proteases of HIV-1 and avian myeloblastosis virus (AMV), respectively, occurs specifically between the p-nitrophenylalanyl and prolyl residues. These hydrolyses are accompanied by an increase in UV absorption at 316 nm, which permits convenient, continuous monitoring of the progress of these reactions. ii) Optical assays for RT and other DNA polymerases have been developed. These assays are based upon changes in circular dichroism (CD) and UV spectra upon elongation of the double helical portion of synthetic template/primer duplexes. In the presence of the Klenow fragment of E. coli DNA polymerase I or HIV-1 RT, incorporation of dTMP into a synthetic template/primer consisting of a 40-60-mer of dA, primed with a 20-mer of dT, produced an enhancement in the negative CD band at 248 nm and a decrease in the UV absorption at 260-275 nm. Both changes are linearly related to the extent of DTMP incorporation. Analogous results were obtained with HIV-1 RT using poly(ra) primed with oligo(dT). These continuous assay methods for retroviral enzymes offer a distinct advantage over single-point assays, in that they permit measurement of the entire time course of the enzymatic reaction using a single 60-120 microliter sample. These methods are presently being employed in studies of the effects of reaction conditions and specific functional-group reagents on the activity of both RT and HIV-1 protease.