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.

通常在稀溶液中研究表征酶催化活性的动力学参数。但是，实际上，这些反应发生在除水和离子之外还充满其他大分子（包括蛋白质，核酸，脂质和代谢物）的细胞中。这种拥挤的环境可能会影响酶催化的反应速率，因此有必要在实验室环境中模仿人群。

我们确定了大分子拥挤者对丙型肝炎病毒蛋白酶NS3 / 4A活性的影响。作为拥挤者，我们使用了聚乙二醇（PEG），Ficoll和牛血清白蛋白。使用带有标记的肽底物的荧光分析，我们发现拥挤物对NS3 / 4A催化反应动力学的影响不同。Ficoll拥挤剂增加，而PEG降低了初始和最大反应速度。为了解释PEG与Ficoll相比所产生的相反作用，我们在显性溶剂中进行了NS3 / 4A的分子动力学模拟，并被其肽底物和PEG分子包围。模拟表明PEG和NS3 / 4A之间的疏水和极性/静电相互作用以及PEG氧和富含氢键供体的NS3 / 4A氨基酸之间形成的氢键。NS3 / 4A蛋白酶是抗病毒药物telaprevir的已知靶标。我们发现Ficoll改变了telaprevir的抑制常数，表明在抑制剂设计中也应考虑拥挤剂的作用。