The Low Barrier Hydrogen Bond in Enzymatic Catalysis

Abstract

The proposal that low barrier (i.e. short, very strong) hydrogen bonds (LBHBs) 1 play a role in enzymatic catalysis was first put forth in 1993 and 1994 (1-4). The proposal was accepted by some but rejected by others (5-8). Initial rejection on theoretical grounds has been followed by increasing experimental support, and recent improvements in theory have been able to account for the experimental observations of LBHBs in enzymes (9 -15). In this minireview we will explain the original proposal, summarize the experimental data from the past few years, and argue that LBHBs do play important roles in enzymatic reactions. Properties of Hydrogen BondsThe strength of a hydrogen bond depends on its length and linearity, the nature of its microenvironment, and the degree to which the pK values of the conjugate acids of the heavy atoms sharing the proton are matched. In water, the hydrogen-bonded oxygens are separated by ϳ2.8 Å, and the ⌬H of formation is ϳ5 kcal mol Ϫ1 . The hydrogen bonds in water are, however, weak because of the poor pK match between the participating oxygen atoms. Because the pKs of H 3 O ϩ and H 2 O are Ϫ1.7 and 15.7, respectively, the proton in the structure H 2 O⅐⅐⅐H-OH is tightly associated with the OH Ϫ group as a water molecule. In the gas phase, where the dielectric constant is low, hydrogen bonds between heteroatoms with matched pKs can be very short and strong, and experimental as well as calculated values of ⌬H of formation can approach 25 or 30 kcal mol Ϫ1 (16, 17). Likewise, in crystals hydrogen bonds can be very strong. The O-O distance in the ion [H- O⅐⅐⅐H⅐⅐⅐O-H] -in a crystal of a chromium complex is only 2.29 Å (18,19). In organic solvents, strong hydrogen bonds can also form, although the ⌬H of formation probably never exceeds 20 kcal mol Ϫ1. Recent calculations suggest that once the dielectric constant is at least 6 the strength of a strong hydrogen bond levels off at a level about half that in the gas phase (13,17). Because the active site of an enzyme is no longer aqueous once it has closed around a substrate, the properties of hydrogen bonds in organic solvents are highly pertinent to enzymatic catalysis.What happens energetically as hydrogen bonds become shortened can be seen in Fig. 1. Structure A represents the situation in water, where the hydrogen is firmly attached to either the lefthand or right-hand oxygen and is more loosely bonded to the other one, with an O-O distance of Ն2.8 Å. There is an energy barrier between the two possible positions of the hydrogen, with the zero point energy levels shown in Fig. 1. Such a hydrogen bond is essentially electrostatic, and the covalent O-H bond is the usual 0.9 -1.0 Å in length.As the overall O-O distance is shortened, the energy barrier drops until it reaches the zero point energy level at an O-O distance of ϳ2.5 Å (Fig. 1B); this is a LBHB. The ⌬H of formation has increased to 15-20 kcal/mol, and the hydrogen can now move freely between the two oxygens. In crystals containing LBHBs, neutron diffraction shows the hydrogen dif...

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