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被用来研究 逆转录酶（RT）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异性抑制剂。 内切酶为了促进这些研究， 已经开发了蛋白酶和RT。i）显色剂的裂解 底物，Ac-Lys-Ala-Ser-Gln-Asn-（对硝基）PhePro-Val-Val-酰胺和 Thr-Phe-Gln-Ala-（对硝基）Phe-Pro-Leu-Arg-Glu-Ala，由 HIV-1和禽成髓细胞瘤病毒（AMV）的逆转录病毒蛋白酶， 分别发生在对硝基苯丙氨酰基和 脯氨酰残基。这些水解伴随着紫外线的增加 在316 nm处的吸收，这允许方便，连续监测 这些反应的进程。ii）RT和其他DNA的光学测定 已经开发了聚合酶。这些测定是基于 双链伸长后的圆二色性（CD）和UV光谱 合成模板/引物双链体的螺旋部分。存在下 E.大肠杆菌DNA聚合酶I或HIV-1 RT，掺入 将dTMP插入由40-60-mer的dA组成的合成模板/引物中， 用20-mer的dT引发，在负CD带中产生增强 在248 nm处的紫外吸收降低，在260-275 nm处的紫外吸收降低。之变动 与DTMP掺入量呈线性相关。类似的结果 用HIV-1 RT，使用用oligo（dT）引发的poly（ra）获得。这些 逆转录病毒酶的连续测定方法提供了明显的优势 在单点测定，因为它们允许测量整个 使用单个60-120微升的酶促反应的时间过程 sample.这些方法目前正用于研究 反应条件和特定官能团试剂对 RT和HIV-1蛋白酶活性。

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.