SUBSTRATE-ANALOG INHIBITION AND ACTIVE-SITE TITRATION OF PURIFIED RECOMBINANT HIV-1 PROTEASE

The aspartyl protease of human immunodeficiency virus 1 (HIV-1) has been expressed in Escherichia coli at high levels, resulting in the formation of inclusion bodies which contain denatured insoluble aggregates of the protease. After solubilization of these inclusion bodies in guanidinium chloride, the protease was purified to apparent homogeneity by a single-step reverse-phase HPLC procedure. The purified, but inactive, protein was denatured in 8 M urea and refolded to produce the active protease. Enzyme activity was demonstrated against the substrate H-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-OH, modeled after the cleavage region between residues 128 and 135 in the HIV gag polyprotein. With this substrate, a Vmax of 1.3 ± 0.2 µmol/(min.mg) and KM of 2.0 ± 0.3 mM were determined at pH 5.5. Pepstatin (Iva-Val-Val-Sta-Ala-Sta-OH) and substrate analogues with the Tyr-Pro residues substituted by Sta, by Pheψ[CH2N]Pro, and by Leuψ[CH(OH)CH2]Val inhibited the protease with K1 values of 360 nM, 3690 nM, 3520 nM, and <10 nM, respectively. All were competitive inhibitors, and the tightest binding compound provided an active site titrant for the quantitative determination of enzymatically active HIV-1 protease. © 1990, American Chemical Society. All rights reserved.