Abstract
Cells are crowded with various macromolecules and metabolites, which affect biochemical reactions in many ways, from the diffusion of substrates to catalytic activities of enzymes. We herein investigated the proteolytic activity of the human immunodeficiency virus type 1 protease (HIV-1 PR) under non-crowded and crowded conditions. The latter environment was mimicked with various (poly)ethylene glycol molecules as crowding agents. We found that these crowding agents affect the kinetic parameters of the HIV-1 PR catalyzed reaction by increasing the Michaelis-Menten constant and decreasing the maximum velocity. The influence of crowding was concentration dependent. We explain this effect by the dynamics of the HIV-1 PR flexible flaps that cover the peptide substrate binding site and are crucial for enzyme activity, and by a possibly slower substrate-enzyme association time in the crowded conditions.