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.
