Computational Evaluations of Charge Coupling and Hydrogen Bonding in the Active Site of a Family 7 Cellobiohydrolase

Abstract

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.

Fungal Cellulases

“…Follow-up work using these tools examined the flexibility of tunnel-enclosing loops as a function of solution pH. 478 The findings of Bu et al were affirmed in that increased loop flexibility with varying pH is the likely source for the pHdependent morphology seen in the neutron scattering studies.…”

The impact of active site protonation on substrate ring conformation in Melanocarpus albomyces cellobiohydrolase Cel7B

“…The fixed protonation state systems were created based on the five most probable protonation states as determined from the CpHMD simulations at pH = 6 ( Table 1). 15 In all protonation schemes the Glu212 remains unprotonated, which is in line with its role as the nucleophilic residue during hydrolysis. 42 The five systems depicting the different protonation schemes (i.e.…”

“…30 To obtain the substrateenzyme complex the approach outlined in our previous work was implemented. 15,17 In brief, a 10-glucose oligomer was used as the flexible ligand and docked to the rigid Ma Cel7B crystal structure using the AutoDock 4.2 software program. 31 The docked conformations with the lowest energies were then compared to the aligned structure of Ma Cel7B and the crystal structure for Trichoderma reesei (Tr) Cel7A (PDB ID: 7CEL), 32 which was co-crystalized with a 9-glucose oligomer in the active site.…”

“…15,17 Those simulations are further evaluated here with respect to the substrate ring conformation in the active site for pH values of 3, 6, and 8. Provided here is only a brief description of the CpHMD simulations since the methodologies 15,17,33 and theoretical background 34,35 have been reported previously. The CpHMD simulation methodology enables the coupling of protein dynamic motions with the ionization state of protonatable amino acids at a given environmental pH all in the framework of a coupled molecular dynamics and Monte Carlo algorithm.…”

“…11 Analyses of the electrostatic energies, 12 threading, 13 and procession 14 of enzyme-substrate systems have led to increased understanding of the kinetic factors of enzymatic digestion. Fully atomistic simulation studies have been used to investigate the flexibility and processivity, [15][16][17] active site pK a values, 15 hydrogen bonding and substrate binding in the catalytic groove, 18,19 product inhibition characteristics, and thermostability 20,21 for several cellulase systems and other related enzymes. 22 Ma Cel7B cellobiohydrolases are known to catalyze hydrolysis by a double displacement net retaining mechanism and therefore the ring conformation at the À1 subsite is instrumental in facilitating the hydrolysis reaction as observed by QM/MM calculations.…”