Crowded environment affects the activity and inhibition of the NS3/4A protease

Abstract

Kinetic parameters characterizing the catalytic activities of enzymes are typically investigated in dilute solutions. However, in reality, these reactions occur in cells that, in addition to water and ions, are full of other macromolecules including proteins, nucleic acids, lipids, and metabolites. Such a crowded environment might affect enzyme-catalyzed reaction rates, so it is necessary to mimic the crowd in laboratory settings. We determined the effect of macromolecular crowders on the activity of the hepatitis C virus protease NS3/4A. As crowders we used polyethylene glycol (PEG), Ficoll, and bovine serum albumin. Using the fluorescence assay with a labeled peptide substrate, we found that the crowders affected the kinetics of the NS3/4A-catalyzed reaction differently. The Ficoll crowders increased and PEG decreased the initial and maximum reaction velocities. To explain the opposite effects exerted by PEG as compared to Ficoll, we performed molecular dynamics simulations of NS3/4A in explicit solvent and surrounded by its peptide substrates and PEG molecules. The simulations suggest both hydrophobic and polar/electrostatic interactions between PEG and NS3/4A with hydrogen bonds formed between PEG oxygens and NS3/4A amino acids rich in hydrogen bonds donors. The NS3/4A protease is a known target for telaprevir, an anti-viral drug. We found that Ficoll changes the inhibition constant for telaprevir suggesting that the effect of crowders should also be considered in inhibitor design.

Keywords: Enzymatic activity; Fluorescence spectroscopy; Hepatitis C virus; Macromolecular crowding; Molecular dynamics simulations; NS3/4A protease.