SUBSTRATE-DEPENDENT MECHANISMS IN THE CATALYSIS OF HUMAN-IMMUNODEFICIENCY-VIRUS PROTEASE

The most preferred residue in the substrates of human immunodeficiency virus (HIV-1) protease is glutamic acid in the P2' position. The cata lytic importance of this charged residue has been studied to obtain a detailed insight into the mechanism of action, which will promote drug design to combat the virus. To this end, we have synthesized Lys-Ala- Arg-Val-LeuPhe(NO2)-Glu-Ala-Nle (substrate E) and its counterpart con taining the neutral Gln (substrate Q) in place of Glu. Kinetic analyse s have shown that the specificity rate constants (k(cat)/K-m) display bell-shaped pH dependencies for both substrates, but the pH-independen t limiting value is 35-40-fold higher with substrate E than with subst rate Q. In contrast to the pH-rate profiles of k(cat)/K-m, there is a striking difference between the pH dependencies of K-m and k(cat) for the two substrates. This indicates different ground state and transiti on state stabilizations in the two reactions. Solvent kinetic deuteriu m isotope effects show that the rate-limiting step for the hydrolysis of substrate E is a chemical step coupled with proton transfer whereas with substrate Q it is a physical step, presumably a conformational c hange. Accordingly, the charged residue in P2' alters the rate-limitin g step and the nature of the enzyme-substrate complex, resulting in di fferent mechanisms for the two substrates.