Enzyme Kinetics Studies of Spin-Labeled Active Human Immunodeficiency Virus Type 1 protease

Abstract

The immunodeficiency virus type 1 protease (HIV-1PR) is a homodimer comprised of 99 amino acid subunits, it functions as an aspartic protease that plays a crucial as role in the maturation of HIV-1. The protease post-translationally cleaves viral polyproteins gag and gag-pol, which makes it a target of AIDS antiviral therapy. The protease has an active site comprised of a D25 residue from each subunit. Accessibility of large, polypeptide substrate to the active site is mediated by a conformational change of two B-hairpin flaps that exposes the active site. The pulsed EPR technique double electron-electron resonance (DEER) has been utilized to measure distances between selected sites on the protease. For spin label incorporation the mutation K55C selected to generate distance profiles. We have shown previously, via traditional SDSL-EPR approaches, that the FDA-approved HIV-1PR inhibitors induce changes in the conformational sampling of the flap regions of the protease. Work described here will determine if spin label incorporation perturbs the kinetics of HIV-1PR constructs analyzed via DEER analysis. Data will ultimately yield rate constants for spin labeled HIV-1PR that will be compared to unlabeled HIV-1PR rate constants to determine if spin label incorporation perturb subtype constructs.