Proton-transfer reactions form an integral part of bioenergetics and enzymatic catalysis. The identification of proton-conducting pathways inside a protein is a key to understanding the mechanisms of biomolecular proton transfer. Proton pathways are modeled here as hydrogen bonded networks of protonconducting groups, including proton-exchanging groups of amino acid side chains and bound water molecules. We focus on the identification of potential proton-conducting pathways inside a protein of known structure. However, consideration of the static structure alone is often not sufficient to detect suitable protontransfer paths, leading, for example, from the protein surface to the active site buried inside the protein.We include dynamic fluctuations of amino acid side chains and water molecules into our analysis. To illustrate the method, proton transfer into the active site of cytochrome P450cam is studied. The cooperative rotation of amino acids and motion of water molecules are found to connect the protein surface to the molecular oxygen. Our observations emphasize the intrinsic dynamical nature of proton pathways where critical connections in the network may be transiently provided by mobile groups.
We investigate the probable proton-transfer pathways from the surface of human carbonic anhydrase II into the active site cavity through His-64 that has been widely implicated as a key residue along the proton-transfer path. A recursive analysis of hydrogen-bonded clusters in the static crystallographic structure shows that there is no complete path through His-64 in either of its experimentally detected conformations. Side chain conformational fluctuation of His-64 from its outward conformation toward the active site is found to provide a crucial dynamic connectivity needed to complete the path coupled to local reorganization of the protein structure and hydration. The energy and free energy barriers along the detected pathway have been estimated to derive the mechanism of His-64 rotation toward the active site. We also investigate a dynamical connectivity map that highlights networks of disordered water molecules that may promote a direct (and probably transient) access of the solvent to the active site. Our studies reveal how such solvent access channels may be related to the putative proton shuttle mediated by His-64. The paths thus identified can be potentially used as reaction coordinates for further studies on the molecular mechanism of enzyme action.
The role of structure and dynamics of an enzyme has been investigated at three different stages of its function including the chemical event it catalyzes. A one-pot computational method has been designed for each of these stages on the basis of classical and/or quantum mechanical-molecular mechanical molecular dynamics and transition path sampling simulations. For a pair of initial and final states A and B separated by a high free-energy barrier, using a two-stage selection process, several collective variables (CVs) are identified that can delineate A and B. However, these CVs are found to exhibit strong cross-coupling over the transition paths. A set of mutually orthogonal order parameters is then derived from these CVs and an optimal reaction coordinate, r, determined applying half-trajectory likelihood maximization along with a Bayesian information criterion. The transition paths are also used to project the multidimensional free energy surface and barrier crossing dynamics along r. The proposed scheme has been applied to the rate-determining intramolecular proton transfer reaction of the well-known enzyme human carbonic anhydrase II. The potential of mean force, F( r), in the absence of the chemical step is found to reproduce earlier results on the equilibrium population of two side-chain orientations of key residue His-64. Estimation of rate constants, k, from mean first passage times for the three different stages of catalysis shows that the rate-determining step of intramolecular proton transfer occurs with k ≃ 1.0 × 10 s, in close agreement with known experimental results.
We report here a transition path sampling study of the conformational fluctuation of His-64 that is known to be important in the enzymatic catalysis of human carbonic anhydrase II. The dynamical transition between experimentally detected conformations of His-64 could not be observed using classical molecular dynamics trajectories extended to 3.5 ns, indicating the transition to be rare on the time scale of molecular dynamics. Using the transition path sampling method, an ensemble of transition paths between these two conformers has been generated and analyzed in detail to identify the mechanism of coupling of His-64 to its neighboring residues during the conformational transition. It is found that both Asn-62 and Tyr-7 may contribute toward retaining the His-64 residue in its outward conformation. Trp-5, on the other hand, shows marked motions at the transition state. The number of water molecules inside a part of the active site cavity and the corresponding cavity volume are also found to vary coupled to the His-64 conformational dynamics.
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