The low-barrier hydrogen bond in enzymic catalysis

W.W. Cleland    Department of Biochemistry and Institute for Enzyme Research, University of Wisconsin-Madison, Madison, WI 53726, USA

Publisher Summary

This chapter describes the various aspects of the low-barrier hydrogen bond in enzymic catalysis. Hydrogen bonds come in a continuum of bond lengths and strengths. The first examples of enzymatic reactions where low-barrier hydrogen bonds played a role involved enolization of the substrate to change the pK of a key group in the reaction. Mandelate racemase enolizes R or S mandelate to convert the carboxyl group into an aci-carboxylate, which can be protonated on opposite sides to give the R or S forms. A low-barrier hydrogen bond forms between Asp102 and His57 in the tetrahedral intermediate of the reaction catalyzed by chymotrypsin and similar serine proteases. Phospholipase A2 catalyzes the hydrolysis of phospholipids at the sn-2 bond, using a water molecule coordinated to Ca2+. Enzymes from bovine pancreas and bee venom are similar in many respects and both contain anaspartate and histidine as catalytic groups. In the presence of phosphonate inhibitors that mimic a tetrahedral intermediate, a low-barrier hydrogen bond exists between the histidine and phosphonate oxygen, whereas the hydrogen bond between the histidine and aspartate is a normal one. A general acid–base catalysis provides a five orders of magnitude rate of acceleration in enzymatic reactions, which is consistent with the energy provided by forming a low-barrier hydrogen bond in the transition state.

1 Introduction

The term “low-barrier hydrogen bond” was introduced by me in 1992 to describe hydrogen bonds between groups of equal pK that showed low deuterium fractionation factors (as low as 0.3).1 It was not until an Enzyme Mechanisms conference in Key Largo, however, that a number of us finally realized how such bonds can help catalyze enzymic reactions and papers describing this appeared in 1993 and 1994.2–5 Since then such bonds have been shown to play a role in many enzymic reactions and a Google search under “low-barrier hydrogen bond” turns up over 5000 hits. In this review I shall describe the properties of low-barrier hydrogen bonds and then give a number of examples. I have not tried to cover the entire literature and apologize to those whose works are not mentioned.

2 Properties of hydrogen bonds

Hydrogen bonds come in a continuum of bond lengths and strengths. Those in water which hold it together as a liquid are ∼2.8 Å between oxygens and are weak (only a few kcal mol−1). Since the pK of water as an acid is above 15 and its pK as a base is less than –1, the pK’s of the two oxygens in the hydrogen bond are drastically different and the hydrogen is covalently bound to one oxygen with a bond distance of 1 Å and weakly bonded electrostatically to the other oxygen. When the pK’s of the two groups are the same, as in a hydrogen bond between formic acid and formate ion, the bond is shorter (2.5–2.6 Å) and the zero point energy level of the hydrogen is at or above the barrier (thus “low-barrier hydrogen bond,” Fig. 1).6–8 Neutron diffraction of crystals containing such bonds show a diffuse distribution centered between the two heavy atoms.9 In certain cases where the bond is especially short, there is no barrier as in the F–H–F− or HO–H–OH− ions which are only 2.3 Å long.10,11 Low-barrier hydrogen bonds are quite strong (as much as 27 kcal mol−1 in the gas phase and perhaps 12 in aqueous solution7), but in a medium with a dielectric constant of ∼7 (similar to what occurs in an enzyme active site) the strength decreases by ∼1 kcal mol−1 per pH unit mismatch in the pK’s of the groups involved.12 Thus there is a continuum between the very strong ones with matched pK’s and the weak ones with very different pK’s and the distances similarly differ as well. Low-barrier hydrogen bonds have considerable covalent character,6,13 which decreases as the bonds weaken and lengthen, so that the weak ones are only electrostatic in nature.

Fig. 1 Energy diagrams for hydrogen bonds between groups of equal pK. (a) Weak hydrogen bond; O–O distance 2.8 Å. (b) Low-barrier hydrogen bond (2.55 Å); the hydrogen diffusely distributed. (c) Single-well hydrogen bond (2.29 Å). Horizontal lines are zero point energy levels for hydrogen (upper) and deuterium (lower).

As noted in 1992, low-barrier hydrogen bonds show low fractionation factors, with up to threefold discrimination against deuterium. They show downfield chemical shifts in proton nuclear magnetic resonance (NMR) of 18–20 ppm. At first it was thought that they only occur in the gas phase or organic solvents, but it is now clear that they can occur in solutions containing a high mole fraction of water, even at room temperature.14,15 What limits their determination in aqueous solution is rapid exchange with solvent protons. Hydrogen bonds can occur between two oxygens, two nitrogens, or one of each. We will show examples of O–O and O–N bonds in the discussion below.

3 Role of low-barrier hydrogen bonds in enzymatic reactions

ENOLIZATION REACTIONS

The first examples of enzymatic reactions where low-barrier hydrogen bonds played a role involved enolization of the substrate to change the pK of a key group in the reaction. Mandelate racemase enolizes R or S mandelate to convert the carboxyl group into an aci-carboxylate which can be protonated on opposite sides to give the R or S forms. In the ground state, one oxygen of the carboxyl group of mandelate is coordinated to Mg2+ and the other oxygen is hydrogen bonded to Glu317 which is protonated.16 The pK of a CO group is low, so this is a weak hydrogen bond. In the aci-carboxylate intermediate, however, the pK of its oxygen will be similar to that of Glu317 and the hydrogen bond becomes a low-barrier one (Fig. 2). The energy liberated by formation of the strong hydrogen bond lowers the activation for formation of the intermediate. The 105 reduction in kcat for the E317Q mutant supports this model.17

Fig. 2 Mechanism of mandelate racemase.16,17 Lys166 and His297 are the two general bases and are on opposite sides of mandelate.

A similar situation occurs with triose-P isomerase, where Glu165 abstracts a proton from either glyceraldehyde-3-P or dihydroxyacetone-P to give an enediolate intermediate. The carbonyl group of the substrate is hydrogen bonded to a neutral imidazole in the active site; this will be a weak hydrogen bond because of the huge mismatch in pK’s.18 The pK of both the imidazole and the enediol intermediate, however will be ∼11, and thus this hydrogen bond becomes a low-barrier one in the intermediate Fig. 3). An isoenergetic shift of the imidazole from one OH to the other shifts the strong hydrogen bond to the oxygen destined to become a carbonyl group when the intermediate is protonated by Glu165 to complete the reaction.

Fig. 3 Mechanism of triose-P isomerase.4 Note the isoenergetic shift of the histidine between the two OH groups of the enediolate intermediate; a low-barrier hydrogen bond is present in both structures.

Ketosteroid isomerase is another enzyme in which enolization of the substrate changes the pK of a key atom so that a low-barrier hydrogen bond forms and helps stabilize the intermediate. Asp38 is the general base that removes a proton from the substrate, and Tyr14 is hydrogen bonded to the carbonyl oxygen of the substrate. The pK’s of a ketone and of tyrosine are drastically different, but in the dienolate intermediate, the pK’s become more similar. An analog aromatic in the A ring and containing a phenolic hydroxyl in place of the ketone bound at least 1000-fold tighter to the D38N mutant than to wild-type isomerase.19 The neutral Asn38 mimics the protonated state of Asp38 after the formation of the intermediate dienolate. In the inhibitor complex proton NMR peaks were at 18.15 and 11.6, with the proton at 18.15 having a deuterium fractionation factor of 0.34 and the hydrogen bond having a strength of 7.1 kcal mol−1 more than one between inhibitor and water. This increase in hydrogen bond strength corresponds to over 5 orders of magnitude rate acceleration and matches the decrease in rate of 4.7 orders of magnitude in the Y14F mutant.

Subsequent work has shown that Asp99 is involved in the hydrogen bond network in this enzyme and the 18.15 ppm NMR peak is from a hydrogen bond between it and Tyr14.20 The 11.6 ppm peak comes from the hydrogen bond between the intermediate and Tyr14. Despite this complexity, it is still true that formation of a strong hydrogen bond in the presence of the intermediate decreases the activation energy of the reaction and thus provides...