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Proc. Natl. Acad. Sci. USA Vol. 95, pp. 12799–12802, October 1998

On the electronic nature of low-barrier bonds in enzymatic reactions

BIRGIT SCHIØTT*, BO BRUMMERSTEDT IVERSEN*†‡,GEORG KENT HELLERUP MADSEN§,FINN KREBS LARSEN§, AND THOMAS C. BRUICE*‡

*Department of , University of California, Santa Barbara, CA 93106; and §Department of Chemistry, University of Aarhus, DK-8000 Århus C, Denmark

Contributed by Thomas C. Bruice, August 11, 1998

ABSTRACT The electronic nature of low-barrier hydro- invoked in the catalytic mechanism. They noted that, when gen bonds (LBHBs) in enzymatic reactions is discussed based known, these enzymes all display the unusually short hetero- on combined low temperature neutron and x-ray diffraction atom distance characterizing SSHB. Furthermore, they sug- experiments and on high level ab initio calculations by using gested that the bonding energy related to formation of an the model substrate benzoylacetone. This has a LBHB could be as high as 10–20 kcal͞mol and that the LBHB LBHB, as the intramolecular hydrogen bond is described by will be essentially covalent (4). Frey et al. (5–7) found evidence, ␦ a double-well potential with a small barrier for hydrogen in the form of very high H, for the likelihood of having an transfer. From an ‘‘atoms in ’’ analysis of the LBHB forming in the of a protease electron density, it is found that the hydrogen atom is stabi- between aspartate and histidinium, which is present in the lized by covalent bonds to both . Large atomic partial tetrahedral intermediate. They suggested that in the Asp-His charges on the hydrogen-bonded atoms are found experimen- LBHB, formal charges of ϽϪ1 and ϩ1 should be assigned to tally and theoretically. Therefore, the hydrogen bond gains Asp and His (5), hereby resulting in a more disperse charge stabilization from both covalency and from the normal elec- distribution in the LBHB relative to a localized Ϫ... ϩ trostatic found for long, weak hydrogen bonds. Asp . H–His form. This lead Warshel et al. (17) to disregard Based on comparisons with other systems having short-strong LBHBs as being important in enzymatic reactions, as they hydrogen bonds or LBHBs, it is proposed that all short-strong proposed that electrostatic interactions alone can explain the and LBHB systems possess similar electronic features of the catalytic power of the enzymatic reactions (18). It should be hydrogen-bonded region, namely polar covalent bonds be- noted that the current generally accepted idea of ‘‘smeared’’ tween the hydrogen atom and both heteroatoms in question. charges associated with LBHBs has so far been based purely on speculations. Kreevoy and Cleland (4) and Frey et al. (5–7) It has been suggested that formation of ‘‘short-strong’’ hydro- suggested that a large degree of covalency will be associated gen bonds (SSHB) or ‘‘low-barrier’’ hydrogen bonds (LBHB) with an enzymatic LBHB, similar to what Gilli et al. (19–24) ␤ can stabilize intermediates and͞or transition states of enzy- have proposed for a series of -keto-. matic reactions (1–13). Originally, the involvement of LBHB In this , we discuss the electronic nature of LBHBs, as in enzymatic reactions was suggested by Cleland (1) based on exemplified by a comparative study of the electron distribution the observation of unusually low hydrogen fractionation fac- of benzoylacetone, which is based on a very low-temperature tors for certain enzymes. He realized that this physicochemical x-ray diffraction study and on a high level ab initio theoretical property can be understood if the potential energy surface of study. the hydrogen bond in question is a double well potential with Ϸ ͞ a low barrier ( 2 kcal mol) toward hydrogen transfer, thus the MODEL AND METHODOLOGY name ‘‘low-barrier hydrogen bond.’’ This description was ␤ adapted from Kreevoy and Liang (15). The idea of having a Benzoylacetone is a -diketone that upon enolization forms a ␤ special type of hydrogen bond in certain enzymatic systems cis- -keto- structure with an intramolecular hydrogen also was invoked by Gerlt and Gassman (2, 3) to explain the bond, which potentially can be either asymmetrical as in 1a and stabilization of various enolic intermediates of enzyme cata- 1b or symmetrical as in 1c (Reaction 1).

lyzed proton abstraction from carbon acids. They proposed a This molecule is a good model substrate for studying the bonding scenario in which the hydrogen resides midway be- electronic nature of a SSHB, as (i) it has a short O⅐⅐⅐⅐⅐O tween the of the enolic intermediate and the conjugate separation of 2.502(4) Å (14), (ii) it resembles the enzymatic base of the catalyst (2). Cleland and Kreevoy (4) enolates where LBHBs have been proposed to form (2–4), and extended the proposal of LBHB as important in enzymatic (iii) in the solid phase, the hydrogen bonded moiety is not systems to cover systems with enolate-types of intermediate

The publication costs of this article were defrayed in part by page charge Abbreviations: SSHB: short-strong hydrogen bond; LBHB: low- barrier hydrogen bond; AIM: atoms in molecules. payment. This article must therefore be hereby marked ‘‘advertisement’’ in †Present address: Department of Chemistry, University of Aarhus, accordance with 18 U.S.C. §1734 solely to indicate this fact. DK-8000 Århus C, Denmark. © 1998 by The National Academy of Sciences 0027-8424͞98͞9512799-4$2.00͞0 ‡To whom correspondence should be addressed. e-mail: bo@kemi. PNAS is available online at www.pnas.org. aau.dk or [email protected].

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involved in other hydrogen bonds (14). The exact position of RESULTS the hydrogen atom of the intramolecular hydrogen bond was The computed theoretical net charges for the hydrogen- determined by low temperature (14). From bonded moiety of benzoylacetone are listed in Table 1 for 1a, these measurements, it was found that the hydrogen is posi- 1b, and 1c. To be able to make comparisons, we have included tioned slightly asymmetric between the two oxygen centers, computed charges for the imaginary non-hydrogen bonded closest to the phenyl-substituted (14), hereby trans-␤-keto-enols, 2a and 2b. These two sets of charges serve establishing the nuclear environment expected for the forma- as reference points for judging the charge redistribution upon tion of an LBHB (2, 4). High level ab initio studies at the ͞ formation of the intramolecular hydrogen bond. Both exper- B3LYP 6–311G(d, p) (26, 27) level of theory confirmed that imental and theoretical atomic partial charges are found in the the intramolecular hydrogen bond is indeed associated with a table for the neutron structure, 3, as measured at 20 K (14). double-well potential having a low barrier toward hydrogen transfer¶ (1.7 kcal͞mol from 1b to 1a, and 2.4 kcal going in the opposite direction) (25), and for example, not a single-well potential as was found theoretically for the formate– (28–31). Structure 1c represents the transition state for the movement of the hydrogen atom from one oxygen center to the other in benzoylacetone. It was found that, upon inclusion of the zero-point vibrational energies, 1c became the lowest energy structure among 1a, 1b, and 1c, and thus it represents the theoretical prediction for the bonding in ben- zoylacetone (25). The theoretical structure is in fine agreement with the low temperature neutron structure (14). Two methods based on analyses of the experimental and theoretical electron distributions were applied to study the electronic nature of the LBHB in benzoylacetone. First, an evaluation of the atomic partial charges of the hydrogen- bonded atoms was carried out. Secondly, Bader’s (32) topo- logical analyses by using the atoms in molecules (AIM) theory were undertaken. The AIM theory is a quantum mechanical theory in which all topological species (atoms, bonds, rings, and cages) that constitute a structure are uniquely defined from the electron density of the molecular system. The struc- tural elements are identified at the critical points of the electron density (ƒ␳ ϭ 0), with atoms being associated to maxima, bonds and rings associated to saddle points, and cages associated to local minima in the electron distribution (32). Bader (32) has shown that the sign of the Laplacian of the electron density at a bond critical point reveals whether charge ƒ2␳ Ͻ is concentrated ( 0) as in a covalent bonds or depleted The results of the AIM-analyses are listed in Table 2 and Table ƒ2␳ Ͼ ( 0) as in electrostatic bonds. Thus, the AIM theory 3 for the electron density, ␳, and the Laplacian, ƒ2␳, respec- provides a tool for characterizing the specific types of bonds tively. Entries for the asymmetric cis-␤-keto-enols (1a and 1b), present in benzoylacetone. for the trans-␤-keto-enols (2a and 2b), the transition state (1c), Experimentally, the electron density is obtained from a low and for the experimental structure (3), with both theoretical temperature (8 K) x-ray diffraction experiment (14). When and experimental values, are listed. using the multipole method (33, 34) for description of the Electronic Nature of an LBHB. The atomic partial charges experimental electron density, atomic partial charges can be reveal that the LBHB in benzoylacetone is a very polar bond estimated from the monopole populations (14). Theoretically, because large partial charges are found (14). Furthermore, it can atomic partial charges are calculated by using the Brenneman be seen in Table 1 that the theoretical and experimental methods et al. (35) scheme for fitting charges to the electrostatic of calculating atomic partial charges give almost quantitative potential at the B3LYP͞6–311G(d, p) level of theory (25). The agreement for the three hydrogen-bonded atoms of the neutron AIM analysis was carried out by using the XD-suite of 3 programs (36) for the experimental electron distribution; structure, , with the theoretical values being numerically slightly whereas for the theoretical analysis, the critical-point option of higher. The theoretical charges indicate that the atomic partial charges of the hydrogen bonded moiety are unaffected by the the AIM-keyword (37–44) as implemented in GAUSSIAN 94 (45) was used. For further details on the analyses, the following ͞ should be consulted (see refs. 14 and 25). Table 1. Atomic charges calculated at the B3LYP 6-311G(d, p) level of theory using Brenneman’s et al. scheme for fitting atomic charges from the electrostatic potential ¶ We use the two terms LBHB and SSHB as follows: The term LBHB Ϫ 2 6 is used when the hydrogen bond is described by a double-well Charge H O O potential energy surface that has a low internal barrier for hydrogen 1a 0.453 Ϫ0.573 Ϫ0.573 transfer (1). An SSHB is any hydrogen bond in which the heteroatom Ϫ Ϫ ⅐⅐⅐⅐⅐ O Ͻ 1b 0.477 0.571 0.611 distance is unusually short, for O H O systems 2.55 Å and for Ϫ Ϫ N⅐⅐⅐⅐⅐HOO Ͻ2.65 Å (13). Several methods for acquiring knowledge 1c 0.490 0.589 0.608 of the shape of the potential energy surface exist, both theoretical and 2a 0.406 Ϫ0.483 Ϫ0.522 experimental. Of the latter, the most notable are spectroscopic 2b 0.327 Ϫ0.403 Ϫ0.527 1 ␦ ⌬ϭ␦ Ϫ ␦ methods (13), such as H NMR (with H and H D as 3 0.451 Ϫ0.559 Ϫ0.593 ␯ ␯ indicators) and IR ( OH and OD) or analysis of the thermal vibrations Experiment 0.40 (3) Ϫ0.51 (5) Ϫ0.45 (4) as measured by low temperature neutron diffraction (14). The special type of hydrogen bond in SSHBs and LBHBs is indicated Experimental charges are included for comparison (14). The atom as, XԽԽԽԽԽԽH, with X being a heteroatom (5–7). numbering scheme corresponds to 1a. Downloaded by guest on September 24, 2021 Biochemistry: Schiott et al. Proc. Natl. Acad. Sci. USA 95 (1998) 12801

Table 2. Electron density at bond critical points found in the keto-enol fragment of molecules 1a, 1b, 1c, 2a, 2b, and 3 ␳ (eÅϪ3)H1–O2 O2–C3 C3–C4 C4–C5 C5–O6 O6–H1 1a 0.419 2.530 1.930 2.171 2.137 2.136 2a n͞a 2.700 1.834 2.236 1.988 2.485 1b 2.102 2.130 2.149 1.946 2.545 0.446 2b 2.475 1.983 2.226 1.823 2.728 n͞a 1c 1.240 2.324 2.050 2.062 2.360 1.160 3 1.084 2.132 2.057 2.026 2.354 0.861 Experiment 0.89 (3) 2.44 (8) 2.17 (4) 2.04 (4) 2.54 (8) 0.76 (3) The experimental value is included for comparison (14). The atom numbering scheme corresponds to 1a.

position of the hydrogen atom between the two oxygens as Cleland (4) and Frey et al. (5–7) proposal of LBHB as being essentially identical charges are computed for 1a, 1b, and 1c; all covalent in enzymatic reactions. The two cis-␤-keto-enol , of which compare well with the charges of the neutron structure, 1a and 1b, both have one covalent and one electrostatic bond, as 3. However, it is interesting to note that the formation of the expected. The emerging description of the electronic nature of hydrogen bond leads to increased charges (compare 1a with 2a the LBHB in benzoylacetone is thus a very polar and 1b with 2b). A consequence of these observations is that as indicated in 4 (25). The strength of the short-strong resonance- LBHBs should not be considered as having more disperse (18) assisted hydrogen bond is estimated to be Ϸ16 kcal͞mol. charge distributions than normal hydrogen bonds. Therefore, this generally accepted assumption is not correct. To learn more about the type of bonds found in the hydrogen bonded portion of benzoylacetone, an AIM analysis was carried out. From the electron densities at the bond critical points of the pseudo 6-membered ring (Table 2), it is again seen that there is essentially quantitative agreement between theory and experiment as very similar numbers are found for 3 (see entry 6 and 7 in Table 2). The theoretically calculated values of the electron densities at the bond critical points all correlate Benzoylacetone represents a globally neutral molecule, as well with the measured bond distances (25); the shorter the does the proposed LBHB between aspartate and histidinium bond, the greater the electron density in the bond. This is in the catalytic triad of serine proteases. Garcia-Viloca et al. especially true for all the oxygen–hydrogen interactions (49) have calculated the electron density and the Laplacian at present in the analyzed molecules. An ordinary OOH bond the two bond critical points found in the transition state for not engaged in hydrogen bonding has a density of 2.5 e͞Å3 (2a hydrogen transfer from 1-methyl imidazolium to 2,2- ͞ ϩ and 2b), whereas an OOH bond engaged in an intramolecular dichloropropionate at the HF 6–31 G(d) level of theory, for hydrogen bond (1a and 1b) has a decreased density of Ϸ2.1 which an LBHB is proposed to form (6). They find numbers ␳ e͞Å3. At the other extreme, normal long hydrogen bonds have very similar to what we find in this study for benzoylacetone [ : ͞ 3 ԽԽԽԽԽԽ 3 ͞ 3 ԽԽԽԽԽԽ ƒ2␳ Ϫ densities of Ϸ0.1 e͞Å3 (46). In between these extremes are the 1.147 e Å (O H) and 0.945 e Å (H N); : 9.881 ͞ 5 ԽԽԽԽԽԽ Ϫ ͞ 5 ԽԽԽԽԽԽ LBHB in 1c and 3 with densities of 0.8–1.2 e͞Å3 s and the short e Å (O H) and 4.338 e Å (H N)], which indicates that asymmetric hydrogen bonds in 1a and 1b with densities of the type of bonds in the two systems are very similar. For a 0.42–0.45 e͞Å3. The observed changes in ␳ upon formation of globally charged system, the LBHB in the maleate ion, Garcia- an asymmetric bond or an LBHB shows that charge redistri- Viloca et al. (50) found properties of the electron density at the bution takes place in the hydrogen-bonded moiety, but as bond critical points of the symmetrically hydrogen-bonded outlined above, this redistribution does not influence the moiety that are very similar to the globally neutral molecules ␳ ϭ ͞ 3 ƒ2␳ ϭϪ ͞ 5 atomic partial charges of the involved atoms. Therefore, ( 1.215 e Å and 10.6 e Å ). This suggests that the although it may be possible to account for the catalytic power electronic nature of a SSHB may be independent of how of enzymes from purely electrostatic considerations (17, 18), it charges are formally assigned for the system—a conclusion is probably not a realistic model due to the observed large that also was reached by Gilli et al. (21) based on a completely degree of charge redistribution in the system (47, 48). different type of analysis. An x-ray diffraction study of the As stated above, the sign of the Laplacian at a bond critical succinate anion, which has a short-strong intermolecular hy- point is a descriptor of the type of bond. The values in Table 3 drogen bond, confirms this statement because properties ␳ ϭ ͞ 3 show that covalent bonds are found between the central hydrogen similar to those of the above molecules ( 1.058 e Å and ƒ2␳ ϭϪ ͞ 5 atom and both oxygens in an LBHB, confirming the Gilli et al. 6.81 e Å ) were found (51). Thus, four chemically (19–24) predictions for ␤-keto-enol systems and Kreevoy and different molecular systems, all possessing a SSHB, show very similar properties of the electron density at the bond critical Table 3. The Laplacian of the electron density at bond critical points of the hydrogen-bonded fragment. points for all hydrogen–oxygen interactions in molecules 1a, 1b, 1c, Implications for Enzymatic LBHB. The above analysis 2a, 2b, and 3 provides evidence for the observation that all SSHB and 2Q (eÅϪ5)H1–O2 O6–H1 LBHB may have very similar characteristics of the electronٌ distribution of the hydrogen-bonded area. Therefore the find- 1a ϩ3.69 Ϫ5.15 ings in the present study may be extrapolated to enzymatic 2a n͞a Ϫ6.12 systems were LBHBs have been suggested. This means that an Ϫ ϩ 1b 5.01 3.72 LBHB in an enzymatic active site can be expected to have Ϫ ͞ 2b 6.13 n a covalent-bonding contributions between the hydrogen and Ϫ Ϫ 1c 8.45 5.65 both heteroatoms involved, as was originally suggested by Ϫ Ϫ 3 6.34 10.91 Cleland and Kreevoy (4) and by Frey et al. (5–7). Our study of Ϫ Ϫ Experiment 9.1 (2) 4.5 (2) benzoylacetone furthermore shows that the hydrogen bonded The experimental values are included for comparison (14). The atoms of SSHB, whether being an asymmetric or close-to- atom numbering scheme corresponds to 1a. , do have large atomic partial Downloaded by guest on September 24, 2021 12802 Biochemistry: Schiott et al. Proc. Natl. Acad. Sci. USA 95 (1998)

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