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

The equilibrium for formation of the intramolecular hydrogen bond (KHB) in a series of substituted salicylate monoanions was investigated as a function of ΔpKa, 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 ΔpKa 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.

Activation of αvβ3-Vitronectin Binding Is a Multistage Process in which Increases in Bond Strength Are Dependent on Y747 and Y759 in the Cytoplasmic Domain of β3

Integrin receptors serve as mechanical links between the cell and its structural environment. Using αvβ3 integrin expressed in K562 cells as a model system, the process by which the mechanical connection between αvβ3 and vitronectin develops was analyzed by measuring the resistance of these bonds to mechanical separation. Three distinct stages of activation, as defined by increases in the αvβ3-vitronectin binding strength, were defined by mutational, biochemical, and biomechanical analyses. Activation to the low binding strength stage 1 occurs through interaction with the vitronectin ligand and leads to the phosphorylation of Y747 in the β3 subunit. Stage 2 is characterized by a 4-fold increase in binding strength and is dependent on stage1 and the phosphorylation of Y747. Stage 3 is characterized by a further 2.5-fold increase in binding strength and is dependent on stage 2 events and the availability of Y759 for interaction with cellular proteins. The Y747F mutant blocked the transition from stage 1 to stage 2, and the Y759F blocked the transition from stage 2 to stage 3. The data suggest a model for tension-induced activation of αvβ3 integrin.

Unusual 1H NMR chemical shifts support (His) Cɛ1—H⋅⋅⋅O⩵C H-bond: Proposal for reaction-driven ring flip mechanism in serine protease catalysis

13C-selective NMR, combined with inhibitor perturbation experiments, shows that the Cɛ1—H proton of the catalytic histidine in resting α-lytic protease and subtilisin BPN′ resonates, when protonated, at 9.22 ppm and 9.18 ppm, respectively, which is outside the normal range for such protons and ≈0.6 to 0.8 ppm further downfield than previously reported. They also show that the previous α-lytic protease assignments [Markley, J. L., Neves, D. E., Westler, W. M., Ibanez, I. B., Porubcan, M. A. & Baillargeon, M. W. (1980) Front. Protein Chem. 10, 31–61] were to signals from inactive or denatured protein. Simulations of linewidth vs. pH demonstrate that the true signal is more difficult to detect than corresponding signals from inactive derivatives, owing to higher imidazole pKa values and larger chemical shift differences between protonated and neutral forms. A compilation and analysis of available NMR data indicates that the true Cɛ1—H signals from other serine proteases are similarly displaced downfield, with past assignments to more upfield signals probably in error. The downfield displacement of these proton resonances is shown to be consistent with an H-bond involving the histidine Cɛ1—H as donor, confirming the original hypothesis of Derewenda et al. [Derewenda, Z. S., Derewenda, U. & Kobos, P. M. (1994) J. Mol. Biol. 241, 83–93], which was based on an analysis of literature x-ray crystal structures of serine hydrolases. The invariability of this H-bond among enzymes containing Asp-His-Ser triads indicates functional importance. Here, we propose that it enables a reaction-driven imidazole ring flip mechanism, overcoming a major dilemma inherent in all previous mechanisms, namely how these enzymes catalyze both the formation and productive breakdown of tetrahedral intermediates.