Proton transport in carbonic anhydrase: Insights from molecular simulation

Abstract

This article reviews the insights gained from molecular simulations of human carbonic anhydrase II (HCA II) utilizing non-reactive and reactive force fields. The simulations with a reactive force field explore protein transfer and transport via Grotthuss shuttling, while the non-reactive simulations probe the larger conformational dynamics that underpin the various contributions to the rate-limiting proton transfer event. Specific attention is given to the orientational stability of the His64 group and the characteristics of the active site water cluster, in an effort to determine both of their impact on the maximal catalytic rate. The explicit proton transfer and transport events are described by the multistate empirical valence bond (MS-EVB) method, as are alternative pathways for the excess proton charge defect to enter/leave the active site. The simulation results are interpreted in light of experimental results on the wild-type enzyme and various site-specific mutations of HCA II in order to better elucidate the key factors that contribute to its exceptional efficiency.

Figure 1

Active site of WT (A), Tyr7Phe (B), and Asn67Leu (C) HCA II, depicting an intramolecular water cluster and critical amino acids. Coordinates were taken from the 2ILI PDB [27], 2NXR PDB [17], and 2NWY PDB [17] structures for the WT, Tyr7Phe, and Asn67Leu, respectively. Figure was rendered with the VMD software [72].

Figure 2

Characteristics of the smallest continuous hydrogen bonded water cluster that connects the zinc-bound water/hydroxide and the protonated N_δ of His64. Water wire size distributions for (A) EZnH₂O²⁺-His in the ‘in’ orientation, (B) EZnH₂O²⁺-His in the ‘out’ orientation, and (C) EZnOH⁺-HisH⁺ in the ‘out’ orientation. Water wire lifetime distributions for (D) EZnH₂O²⁺-His in the ‘in’ orientation, (E) EZnH₂O²⁺-His in the ‘out’ orientation, and (F) EZnOH⁺-HisH⁺ in the ‘out’ orientation. All plots have data from the WT (no fill), Tyr7Phe (light grey fill), and Asn67Leu (dark grey fill) HCA II.

Figure 3

Various computed free energy curves (PMFs) for the proton translocation in the HCA II enzyme. The PMFs are plotted as a function of the radial distance of the CEC to the catalytic zinc, R_Zn-CEC, for the MS-EVB simulation. The SCC-DFTB simulation reaction coordinate (originally a value of zero to one) was converted to R_Zn-CEC based on the location of the zinc-bound solvent minimum and the His64H⁺ minimum (depending on the orientation of His64). The SCC-DFTB simulations are for their proposed ‘proton hole’ hydroxide transfer mechanism [35], while the MS-EVB simulations represent a hydrated excess proton transfer [33]. Also shown are the SCC-DFTB His64Ala mutant PMF results for hydrated excess proton transport.[35]

Figure 4

Spatial occupancy density plot of the excess proton CEC superimposed on the x-ray structure of the WT HCA II enzyme (2CBA). The grey volume represents the isosurface within which the excess proton CEC resides 98% of the time.