The change in hydrogen bond strength accompanying charge rearrangement: implications for enzymatic catalysis

Abstract

The equilibrium for formation of the intramolecular hydrogen bond (KHB) in a series of substituted salicylate monoanions was investigated as a function of delta pKa, the difference between the pKa values of the hydrogen bond donor and acceptor, in both water and dimethyl sulfoxide. The dependence of log KHB upon delta pKa is linear in both solvents, but is steeper in dimethyl sulfoxide (slope = 0.73) than in water (slope = 0.05). Thus, hydrogen bond strength can undergo substantially larger increases in nonaqueous media than aqueous solutions as the charge density on the donor or acceptor atom increases. These results support a general mechanism for enzymatic catalysis, in which hydrogen bonding to a substrate is strengthened as charge rearranges in going from the ground state to the transition state; the strengthening of the hydrogen bond would be greater in a nonaqueous enzymatic active site than in water, thus providing a rate enhancement for an enzymatic reaction relative to the solution reaction. We suggest that binding energy of an enzyme is used to fix the substrate in the low-dielectric active site, where the strengthening of the hydrogen bond in the course of a reaction is increased.

Scheme I

Scheme II

Figure 1

The dependence of log K^HB upon ΔpK_a^water in DMSO (○) and H₂O (□) for the H bond in substituted SA monoanions. The Brønsted slopes are 0.05 and 0.73 in water and DMSO, respectively. A common ΔpK_a scale in water was used to allow a direct comparison of the magnitude of changes in H bond strength in the two solvents. Data are from Table 1.

Figure 2

A greater increase in H bond strength accompanying changes in the charge density of donor/acceptors in DMSO than in water. The logarithm of equilibrium constants for H bonding in DMSO are plotted against those in water for substituted salicylates (note the difference in scales). The solid line represents a least squares fit of the data and gives a slope of 15. The dashed line with a slope of unity, which represents the correlation expected if the strengthening of the H bond were the same in the two media, is shown for comparison. Data are from Table 1.

Figure 3

Diagrammatic illustration of potential catalysis that can be obtained from a greater strengthening of H bonds accompanying charge rearrangements at a nonaqueous enzymatic active site than in water, using the example of the TIM reaction. The pK_a of the substrate carbonyl oxygen increases during the course of reaction, so that ΔpK_a between this oxygen atom and the enzymatic or solution H bond donor (His-E or HOH, respectively) decreases. This leads to an increase in H bond strength. This increase would be greater at the enzymatic active site (ΔΔG^E) than in solution (ΔΔG^soln) because of the greater Brønsted slopes in nonaqueous environments. The amount of catalysis obtained from this strategy, relative to the solution reaction, is ΔΔG^≠ = ΔΔG^E − ΔΔG^soln.

Figure 4

A large Brønsted slope for H bonding in proteins is inferred from folding studies of Staphylococcal nuclease mutants. (A) Thermodynamic analysis depicting the effect of changing the strength of the H bond donor, the substituted tyrosine hydroxyl, on the stability of the protein. The folding equilibrium of a hypothetical non-H-bonded species (K_folding^{no HB}) is used to dissect the effects from H bonding. (B) Schematic depiction of the dependences of H bonding and folding equilibria on the pK_a value of substituted tyrosines. As the tyrosine hydroxyl becomes more acidic, the strengthening of its H bond to the enzymatic glutamate and to water stabilizes the folded and unfolded protein, respectively. The slope of the plot of log K_folding^obsd versus pK_a is the difference between the slopes of plots of log K_E^HB versus pK_a and K_water^HB versus pK_a. This follows from the thermodynamic relationship shown in A, which gives Δlog K_folding^obsd = Δlog K_E^HB − Δlog K_water^HB (i.e., the greater strengthening of the H bond on the protein results in a change in the observed stability of the protein. Note that Δlog K_folding^{no HB} = 0 by definition because the folding equilibrium between the non-H-bonded species does not depend on the strength of the H bond donor.