Hydration of enzyme in nonaqueous media is consistent with solvent dependence of its activity - PubMed (original) (raw)

Comparative Study

Hydration of enzyme in nonaqueous media is consistent with solvent dependence of its activity

Lu Yang et al. Biophys J. 2004 Aug.

Abstract

Water plays an important role in enzyme structure and function in aqueous media. That role becomes even more important when one focuses on enzymes in low water media. Here we present results from molecular dynamics simulations of surfactant-solubilized subtilisin BPN' in three organic solvents (octane, tetrahydrofuran, and acetonitrile) and in pure water. Trajectories from simulations are analyzed with a focus on enzyme structure, flexibility, and the details of enzyme hydration. The overall enzyme and backbone structures, as well as individual residue flexibility, do not show significant differences between water and the three organic solvents over a timescale of several nanoseconds currently accessible to large-scale molecular dynamics simulations. The key factor that distinguishes molecular-level details in different media is the partitioning of hydration water between the enzyme and the bulk solvent. The enzyme surface and the active site region are well hydrated in aqueous medium, whereas with increasing polarity of the organic solvent (octane --> tetrahydrofuran --> acetonitrile) the hydration water is stripped from the enzyme surface. Water stripping is accompanied by the penetration of tetrahydrofuran and acetonitrile molecules into crevices on the enzyme surface and especially into the active site. More polar organic solvents (tetrahydrofuran and acetonitrile) replace mobile and weakly bound water molecules in the active site and leave primarily the tightly bound water in that region. In contrast, the lack of water stripping in octane allows efficient hydration of the active site uniformly by mobile and weakly bound water and some structural water similar to that in aqueous solution. These differences in the active site hydration are consistent with the inverse dependence of enzymatic activity on organic solvent polarity and indicate that the behavior of hydration water on the enzyme surface and in the active site is an important determinant of biological function especially in low water media.

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Figures

FIGURE 1

MD snapshot of the surfactant-solubilized subtilisin BPN′ in OCT: the enzyme (green), water molecules (red and white), surfactant AOT (cyan), and some of the surrounding OCT (white wireframe) molecules are shown. The cubic simulation box is bigger than the image shown, and the space surrounding the enzyme is filled with solvent; only a layer of solvent molecules is shown, however, for visual clarity. The active site residues are highlighted (blue spacefill).

FIGURE 2

Analysis of the enzyme flexibility from MD simulation trajectories: (a) time-averaged backbone structures of subtilisin in water (red), OCT (green), THF (blue), and ACN (magenta) superimposed onto the backbone of crystal structure of subtilisin in water (yellow), (b) instantaneous RMSD of the enzyme backbone in a given solvent from the time-averaged structure in the same solvent, and (c) time-averaged RMSD of residue heavy atoms with respect to their location in the average enzyme structure.

FIGURE 3

Water dynamics in organic media (OCT, THF, and ACN) obtained from MD simulations. Water molecules (wireframe red and white) in 90 equally spaced configurations are shown; rigid body rotations and translations were applied such that enzyme backbones (not shown) are superimposed in these snapshots. Based on their dynamics, water molecules can be classified into three categories: tightly bound (red spacefill), weakly bound (blue spacefill), and mobile (yellow spacefill). Only one example (out of many) of each of these three types of water molecules is highlighted in the figure. Although water molecules that are stripped away from the enzyme are always mobile, mobile water molecules can also be found on the enzyme surface (top left). The bottom right panel shows the number of water molecules within 4 Å of the enzyme, the so-called biological water, in different solvents as a function of time. Horizontal lines show the time-averaged values.

FIGURE 4

Solvent penetration and the active site hydration in different solvents. (a) Spherically averaged number densities of organic solvent heavy atoms normalized by their bulk density, g(r) = ρ(r)/_ρ_bulk, as a function of distance, r, from the enzyme center. The appropriately scaled density of the enzyme heavy atoms (black line) is shown for reference. (b) Spherically averaged number densities of water molecules (_ρ_water(r), in units of molecules/Å3) as a function of distance (in Å units) from the enzyme center. c shows the detailed three-dimensional density maps of different molecules in the vicinity of the active site. The enzyme backbone is shown by ribbons, whereas the catalytic triad residues, Asp-32, His-64, and Ser-221, are identified separately in a wireframe representation. The oxygen atom of Ser-221 is colored green to distinguish it from the vicinal density map. The ensemble-averaged densities of water (blue) and organic solvent (red) are shown by spheres of radii proportional to the local density at a grid point. Thus, the large blue spheres indicate tightly bound structural water molecules, whereas uniformly spread smaller blue spheres indicate the weakly bound or mobile water molecules in the active site region.

FIGURE 5

Representative MD snapshot of the active site of subtilisin in ACN. The catalytic triad and Asn-155 of the oxyanion hole are shown with sticks, and the ACN molecules are shown with balls-and-sticks (carbon, light green; oxygen, red; nitrogen, blue; and hydrogen, white). The van der Waals surface (generated by VMD; Humphrey et al., 1996) of the protein in the active site region is shown in gray.

FIGURE 6

The time-dependent distances between heavy atoms involved in hydrogen bonds HB1 and HB2; from Ser-221 O_γ_atom to His-64 N_ε_ atom (red) and from His-64 N_δ_ to Asp-32 O (green) in different organic solvents and in water.

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Hydration of enzyme in nonaqueous media is consistent with solvent dependence of its activity - PubMed (original) (raw)