Computational evaluations of charge coupling and hydrogen bonding in the active site of a family 7 cellobiohydrolase.

Solution pH and the pKa values of ionizable residues are critical factors known to influence enzyme catalysis, structural stability, and dynamical fluctuations. Presented here is an exhaustive computational study utilizing long time constant pH molecular dynamics, pH replica exchange simulations, and kinetic modeling to evaluate pH-dependent conformations, charge dynamics, residue pKa values, and the catalytic activity-pH profile for cellobiohydrolase Cel7B from Melanocarpus albomyces . The predicted pKa values support the role of Glu212 as the catalytic nucleophile and Glu217 as the acid-base residue. The presence of a charge-correlated active site and an extensive hydrogen bonding network is found to be critical in enabling favorable residue orientations for catalysis and shuttling excess protons around the active site. Clusters of amino acids are identified that act in concert to effectively modulate the optimal pH for catalysis while elevating the overall catalytic rate with respect to a noncoupled system. The work presented here demonstrates the complex and critical role of coupled ionizable residues to the proper functioning of cellobiohydrolase Cel7B, functionally related glycosyl hydrolases, and enzymes in general. The simulations also support the use of the CpHMD for the accurate prediction of residue pKa values and to evaluate the impact of pH on protein structure and charge dynamics.