Surveying Enzyme Crystal Structures Reveals the Commonality of Active-Site Solvent Accessibility and Enzymatic Water Networks

Abstract

Despite the demonstrable dependence of enzyme functionality on solvation, the notion of water being directly chemically required for catalysis inside active sites remains unexplored. Here we report that over 99% of 1013 enzyme crystals obtained by X-ray crystallography with high resolution (<1.5 Å) contain continuous chains of water linking residues within the active site to bulk water. Also reported are the findings which inspired this study-that electric fields experienced by water hydrogen atoms are on average twice as strong in the active sites of both chains of bacterial polynucleotide kinase (PDB 4QM6) structures compared to those in bulk water. These results point to the possibility that water molecules within active sites may be paramount to the immense catalytic power of enzymes, especially for mechanisms requiring hydronium or hydroxide ions.

Figure 1

(Left): Graphic depictions of the solvation process of enzymes used in this work. Here the red dots are O atoms labeled as HOH in the raw PDB file. (Right): Schematic diagram depicting the procedure for solvation of enzyme PDB files by PACKMOL.

Figure 2

An illustrated example of elucidating a water wire for the active site of triose phosphate isomerase (XRC crystal structure resolution: 1.2 Å, PDB code: 1NEY). The atoms of active site residues Glu 165 and His 95 can be seen as red spheres, the enzyme structure as a green cartoon depiction, and the water atoms belonging to the water wire as yellow spheres. (a) shows the structure before probing for water near the active site residues. (b) shows water molecules within 5 Å of active site atoms. (c), (d), (e), (f) show the recursive expansion of the water wire by adding water molecules whose oxygens are within 5 Å of the current water wire oxygen atoms. In (e) and (f), the water wire has reached the bulk solvent and can be seen extending far from the enzyme. By this point, the wire-generating algorithm would stop as the solvent has been contacted.

Figure 3

A histogram contrasting the exceptionally high electric fields felt by the O2’–H2’ bond of the RG nucleotide in the active sites of the C and D chains of bacterial polynucleotide kinase PDB 4QM6(24) to the fields on liquid water.

Figure 4

A histogram of the exceptionally high electric fields felt by water molecules near the active site bound GTP (red circles) and phosphate (blue triangles) in the bacterial polynucleotide kinase PDB 4QM6, and in bulk water (black squares). The presence of Mg2+ ions likely contributes to the strong fields.

Figure 5

Histograms of water molecules per water wire originating from the active site (solid black line) and two random adjacent residues (dashed line) in each of 1013 crystal structures. Each water wire was grouped by the criterion of r(O–O) < 5 Å. Relative frequencies were calculated as the number of structures with their water wire in the range of each bin, divided by the total number of structures. These are smoothed histograms with bin size 50.

Figure 6

By the basis of enzyme class, histograms of water molecules per wire originating from the active site among 1013 enzyme crystal structures. Each water wire was grouped by the criterion of r(O–O) < 5 Å and required to have one member whose oxygen was this distance from any active site atom. Relative frequencies were calculated as the number of structures with their water wire in the range of each bin, divided by the total number of structures. These are smoothed histograms with bin size 50.

Figure 7

Oxygen–oxygen distances between each water molecule and its nearest neighboring water molecule within the active site-contiguous water wires of 1013 enzyme structures. Relative frequencies were calculated as the number of structures with their water wire in the range of each bin, divided by the total number of structures. These are smoothed histograms with bin size 50.