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

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

Shu-ou Shan and
Daniel Herschlag†

Department of Biochemistry, B400 Beckman Center, Stanford University, Stanford, CA 94305-5307

Abstract

The equilibrium for formation of the intramolecular hydrogen bond (K^HB) in a series of substituted salicylate monoanions was investigated as a function of ΔpK_a, the difference between the pK_a values of the hydrogen bond donor and acceptor, in both water and dimethyl sulfoxide. The dependence of log K^HB upon ΔpK_a 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.

Footnotes

↵ † To whom reprint requests should be addressed.
John I. Brauman, Stanford University, Stanford, CA
Abbreviations: DMSO, dimethyl sulfoxide; TIM, triosephosphate isomerase; SA, salicylic acid; LBHB, low-barrier H bonds.
↵ ‡ H bonding between the COOH and OH groups may also be present in the neutral acids. This would increase the observed pK_a values of the carboxylic acids, leading to underestimate of the strength of the H bond in the monoanion. This effect is expected to be small, however, because the H bonds involving neutral species in the acid form are generally much weaker than those involving charged species in the anionic form (10–14, 16–25).
↵ § The steeper dependence of log K^HB upon ΔpK_a ^water in DMSO can be broken down into two components. (i) The pK_a scale in DMSO is expanded: for ionization of phenols and benzoic acids, a change in ΔpK_a of 1 in water corresponds to a change in ΔpK_a of ≈2.4 in DMSO (Table 1 and refs. 2–4). (ii) A steeper dependence of H bond strength on the proton affinity of the donor and acceptor: a plot of log K^HB versus ΔpK_a ^DMSO for the H bond in salicylates yields a Brønsted slope of 0.30 (not shown), whereas the slope is 0.05 for the corresponding H bonds in water (Fig. 1).
↵ ¶ The Brønsted slope of 0.05 for H bonding in SA monoanions in water is similar to the value of 0.04–0.05 calculated from the Hine equation (Eq. 3), using a τ value of 0.01 in water (11–14). $\text{[math]}$ {altfoot}This equation is based on a electrostatic model of H bonding and describes H bonding in water as the competition between H bonding between solutes versus H bonding to water (11–14).
↵ ‖ The observed stability of the proteins at pH 7.0 as a function of the aqueous pK_a of substituted tyrosines gives a Brønsted slope of 0.35 (37). The actual Brønsted slope for the H bond is likely to be larger because tetrafluorotyrosine is predominantly deprotonated in solution at pH 7.0 [pK_a ≈ 5.3 (37)]. The folded protein with the deprotonated tetrafluorotyrosine is expected to be less stable than those with the protonated form because of electrostatic repulsion between the anionic glutamate and hydroxylate of tetrafluorotyrosine. This presumably leads to an underestimate of the stability of the protein with the neutral tetrafluorotyrosine. Correcting for the fraction of deprotonated species at pH 7.0, assuming that all of the folded proteins contain the protonated tetrafluorotyrosine, yields a Brønsted slope of 0.75. This provides an estimate for the upper limit of the Brønsted slope.