Role of charged residues in the catalytic mechanism of hepatitis C virus NS3 protease: Electrostatic precollision guidance and transition-state stabilization

Maturational cleavage of the hepatitis C virus polyprotein involves the vir al chymotrypsinlike serine protease NS3. The substrate binding site of this enzyme is unusually flat and featureless. We here show that NS3 has a high ly asymmetric charge distribution that is characterized by strong positive potentials in the vicinity of its active site and in the S5/S6 region. Usin g electrostatic potential calculations, we identified determinants of this positive potential, and the role of six different residues was explored by site-directed mutagenesis. Mutation of residues in the vicinity of the acti ve site led to changes in k(cat) values of a peptide substrate indicating t hat basic amino acids play a role in the stabilization of the transition st ate. Charge neutralization in the S5/S6 region increased the K-m values of peptide substrates in a manner that depended on the presence of negatively charged residues in the P5 and P6 positions. K-i values of hexapeptide acid s spanning P6-P1 (product inhibitors) were affected by charge neutralizatio n in both the active site region and the S5/S6 region. pre-steady-state kin etic data showed that the electrostatic surface potential is used by this e nzyme to enhance collision rates between peptidic ligands and the active si te. Calculations of the interaction energies of protease-substrate or prote ase-inhibitor complexes showed that electrostatic interaction energies oppo se the formation of a tightly bound complex due to an unfavorable change in the desolvation energy. We propose that desolvation costs are minimized by avoiding the formation of individual ion pair interactions through the use of clusters of positively charged residues in the generation of local elec trostatic potentials.