The Low Barrier Hydrogen Bond in Enzymatic Catalysis

low barrier hydrogen bond. 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 (1Gerlt J.A. Gassman P.G. Biochemistry. 1993; 32: 11934-11952Crossref PubMed Scopus (376) Google Scholar, 2Gerlt J.A. Gassman P.G. J. Am. Chem. Soc. 1993; 115: 11552-11568Crossref Scopus (386) Google Scholar, 3Cleland W.W. Kreevoy M.M. Science. 1994; 264: 1887-1890Crossref PubMed Scopus (1062) Google Scholar, 4Frey P.A. Whitt S. Tobin J. Science. 1994; 264: 1927-1930Crossref PubMed Scopus (739) Google Scholar). The proposal was accepted by some but rejected by others (5Warshel A. Papazyan A. Kollman P.A. Science. 1995; 269: 102-104Crossref PubMed Scopus (334) Google Scholar, 6Warshel A. Papazyan A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13665-13670Crossref PubMed Scopus (186) Google Scholar, 7Guthrie J.P. Chem. Biol. 1996; 3: 163-170Abstract Full Text PDF PubMed Google Scholar, 8Scheiner S. Kar T. J. Am. Chem. Soc. 1995; 117: 6970-6975Crossref Scopus (135) Google Scholar). 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 (9Gerlt J.A. Kreevoy M.M. Cleland W.W. Frey P.A. Chem. Biol. 1997; 4: 259-267Abstract Full Text PDF PubMed Scopus (335) Google Scholar, 10Pudzianowski A.T. Recent Res. Devel. Physical Chem. 1997; 1: 81-97Google Scholar, 11Pan Y. McAllister M.A. J. Am. Chem. Soc. 1997; 119: 7561-7566Crossref Scopus (85) Google Scholar, 12Pan Y. McAllister M.A. J. Org. Chem. 1997; 62: 8171-8176Crossref PubMed Scopus (40) Google Scholar, 13Pan Y. McAllister M.A. J. Am. Chem. Soc. 1998; 120: 166-169Crossref Scopus (83) Google Scholar, 14Smallwood C.J. McAllister M.A. J. Am. Chem. Soc. 1997; 119: 11277-11281Crossref Scopus (72) Google Scholar, 15Kumar G.A. McAllister M.A. J. Am. Chem. Soc. 1998; 120: 3159-3165Crossref Scopus (62) Google Scholar). 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. The 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 ∼2.8 Å, and the ΔH of formation is ∼5 kcal mol−1. The hydrogen bonds in water are, however, weak because of the poor pK match between the participating oxygen atoms. Because the pKs of H3O+ and H2O are −1.7 and 15.7, respectively, the proton in the structure H2O···H–OH is tightly associated with the OH− 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 ΔH of formation can approach 25 or 30 kcal mol−1 (16Garcia-Viloca M. Gonzalez-Lafont A. Lluch J.M. J. Am. Chem. Soc. 1997; 119: 1081-1086Crossref Scopus (144) Google Scholar, 17McAllister M.A. Can. J. Chem. 1997; 75: 1195-1202Crossref Scopus (65) Google Scholar). Likewise, in crystals hydrogen bonds can be very strong. The O–O distance in the ion H–O···H···O–H– in a crystal of a chromium complex is only 2.29 Å (18Abu-Dari K. Raymond K.N. Freyberg D.P. J. Am. Chem. Soc. 1979; 101: 3688-3689Crossref Scopus (92) Google Scholar, 19Abu-Dari K. Freyberg D.P. Raymond K.N. Inorg. Chem. 1979; 18: 2427-2433Crossref Scopus (29) Google Scholar). In organic solvents, strong hydrogen bonds can also form, although the ΔH of formation probably never exceeds 20 kcal mol−1. 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 (13Pan Y. McAllister M.A. J. Am. Chem. Soc. 1998; 120: 166-169Crossref Scopus (83) Google Scholar, 17McAllister M.A. Can. J. Chem. 1997; 75: 1195-1202Crossref Scopus (65) Google Scholar). 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 Arepresents the situation in water, where the hydrogen is firmly attached to either the left-hand or right-hand oxygen and is more loosely bonded to the other one, with an O–O distance of ≥2.8 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 ∼2.5 Å (Fig. 1 B); this is a LBHB. The ΔH 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 diffusely distributed with its average position in the center (20Steiner T. Saenger W. Acta Crystallogr. Sect. B Struct. Sci. 1994; 50: 348-357Crossref Scopus (261) Google Scholar). LBHBs are largely covalent (21Gilli P. Bertolase V. Ferretti V. Gilli G. J. Am. Chem. Soc. 1994; 116: 909-915Crossref Scopus (1053) Google Scholar). There are a number of possible structures between A and B in Fig. 1; however, the covalent O–H bond becomes longer and the overall covalent character of the hydrogen bond increases as the hydrogen bond becomes shorter and stronger (20Steiner T. Saenger W. Acta Crystallogr. Sect. B Struct. Sci. 1994; 50: 348-357Crossref Scopus (261) Google Scholar). Further shortening leads to the limit of 2.29 Å and structure C in Fig. 1. The LBHB has other physiochemical properties in addition to its short heteroatom distance. Its proton NMR chemical shift is far downfield (17–21 ppm), and it can be observed in aqueous solution by application of appropriate water suppression pulse sequences when the exchange rate is slower than the spectrometer frequency. A second property is the low deuterium fractionation factor. Deuterium becomes enriched in more stiffly bonded positions, and the fractionation factor measures the degree of discrimination against deuterium in a given bond relative to the OH bonds in water. Because the covalent O–H distance increases from ∼1.0 to ∼1.2 Å as a weak hydrogen bond is converted into a LBHB, the bond order decreases and thus the discrimination against deuterium increases. Fractionation factors as low as 0.3 have been measured for symmetrical LBHBs such as that betweenp-nitrophenol and p-nitrophenolate ion in organic solvents (22Kreevoy M.M. Liang T.M. J. Am. Chem. Soc. 1980; 102: 3315-3322Crossref Scopus (181) Google Scholar). The fractionation factor of the hydrogen in a LBHB in a protein can be measured by integrating the low field proton NMR peak in mixed H2O/D2O mixtures. Other spectroscopic properties of LBHBs that have been used in studies of small molecules include perturbations of IR stretching frequency and differences in proton and deuterium NMR chemical shifts. For a review of the properties of hydrogen bonds, including LBHBs, see Ref. 23Hibbert F. Emsley J. Adv. Phys. Org. Chem. 1990; 26: 255-379Google Scholar. Although the existence of LBHBs has been known to physical organic chemists for many years, the presence of LBHBs in proteins and the way in which they could play a role in enzymatic catalysis by conversion of a weak hydrogen bond in the initial enzyme-substrate complex into a LBHB in the transition state was only recently recognized (1Gerlt J.A. Gassman P.G. Biochemistry. 1993; 32: 11934-11952Crossref PubMed Scopus (376) Google Scholar, 2Gerlt J.A. Gassman P.G. J. Am. Chem. Soc. 1993; 115: 11552-11568Crossref Scopus (386) Google Scholar, 3Cleland W.W. Kreevoy M.M. Science. 1994; 264: 1887-1890Crossref PubMed Scopus (1062) Google Scholar, 4Frey P.A. Whitt S. Tobin J. Science. 1994; 264: 1927-1930Crossref PubMed Scopus (739) Google Scholar). When a mechanism involves formation of an unstable intermediate, the transition state for forming it will closely resemble it, and the LBHB will also be found in the intermediate or in an enzyme-inhibitor complex that closely mimics it. Such mimics of metastable intermediates at enzymatic sites allow observation of LBHBs by spectroscopic and crystallographic methods (4Frey P.A. Whitt S. Tobin J. Science. 1994; 264: 1927-1930Crossref PubMed Scopus (739) Google Scholar, 24Cassidy C.S. Lin J. Frey P.A. Biochemistry. 1997; 36: 4576-4584Crossref PubMed Scopus (174) Google Scholar). An enzyme converts a weak hydrogen bond into a strong one by changing the pK value of the substrate so that it is close to that of the enzymatic group to which it is hydrogen bonded. The pKvalues for protonating ketones are approximately −5, whereas the pK values of the corresponding enols or enediols are 10–14. If the enzymatic group hydrogen-bonded to the ketone is a neutral imidazole ring of a histidine (pK 14 for imidazolide formation), there will be a good pK match between the enolate and histidine, though not between the ketone and histidine. The increased strength of the LBHB between the enolate and neutral histidine can provide much of the energy needed to bring about enolization of the substrate (a Brønsted base is, of course, also required). One criticism leveled at this proposal was that hydrogen bond strengths did not depend so heavily on pK matching (8Scheiner S. Kar T. J. Am. Chem. Soc. 1995; 117: 6970-6975Crossref Scopus (135) Google Scholar, 25Shan S. Loh S. Herschlag D. Science. 1996; 272: 97-101Crossref PubMed Scopus (260) Google Scholar). However, the data of Shan and Herschlag (25Shan S. Loh S. Herschlag D. Science. 1996; 272: 97-101Crossref PubMed Scopus (260) Google Scholar, 26Shan S. Herschlag D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14474-14479Crossref PubMed Scopus (199) Google Scholar) show that the variation in ΔG of formation with ΔpK depends on the solvent. Although the slope of a plot of the log of the formation constant (K form) versusΔpK is only 0.05 in water, it is 0.73 in dimethyl sulfoxide for a series of salicylate monoanions and 0.65 in tetrahydrofuran for complexes between phenols of varying pK. Enzyme active sites are non-aqueous, and the effective dielectric constants resemble those in organic solvents rather than that in water. If we assume a slope of 0.7, this corresponds to a weakening of the hydrogen bond by a factor of 5 for each unit mismatch in pKvalues. If this change pertains over the full range of pKvalues, a change of −5 to 14 in pK would provide over 18 kcal mol−1 of energy, which corresponds to over 13 orders of magnitude in rate acceleration. Although the slope of log(K form) versus ΔpKprobably does not remain constant as the ΔpK increases, it is clear that considerable energy is potentially available to help catalyze enzymatic reactions. Enzymes catalyze the removal or addition of protons to substrates by employing their side chain functional groups as Brønsted acid or base catalysts. In the reaction catalyzed by lactate dehydrogenase, proton transfer from the OH group of lactate to His-195 accompanies hydride transfer to NAD. The pK of lactate is ∼15, whereas that of pyruvate is approximately −5 and the pK of the histidine imidazolium group is ∼6. Therefore, the hydrogen bond between lactate and His-195 is weak, with a pK mismatch of ∼9 pH units. Likewise, the hydrogen bond between pyruvate and protonated His-195 has a mismatch of ∼11 units. During the reaction the pK of the lactate crosses that of His-195, and at that point the match in pK values should lead to strengthening of the hydrogen bond, accompanied by lowering of the activation energy for the reaction. The elimination of a pK mismatch of 9 units could potentially accelerate the reaction by 4.5 orders of magnitude, which is a typical contribution of general acid/base catalysis to the rate of an enzymatic reaction (the factor can reach 105(27Meloche H.P. O'Connell E.L. J. Protein Chem. 1983; 2: 399-410Crossref Scopus (5) Google Scholar)). A clear example of a LBHB in enzymatic catalysis is shown by chymotrypsin. The low field proton seen at δ 18.3 ppm for the proton between His-57 and Asp-102 in acidic solutions of chymotrypsin (28Robillard G. Shulman R.G. J. Mol. Biol. 1972; 71: 507-511Crossref PubMed Scopus (143) Google Scholar) has been assigned as a LBHB (4Frey P.A. Whitt S. Tobin J. Science. 1994; 264: 1927-1930Crossref PubMed Scopus (739) Google Scholar). Chemical models in nonaqueous media confirm the low field NMR signal, as well as reveal characteristic Fourier transfer IR features (29Tobin J.B. Whitt S.A. Cassidy C.S. Frey P.A. Biochemistry. 1995; 34: 6919-6924Crossref PubMed Scopus (94) Google Scholar). The LBHB-containing structureI (Scheme FSI) displays the low field proton signal as well as a very short N–O distance of y ≥ 0.5. The LBHB-containing complex I is related to the transition state or tetrahedral intermediate for the formation of acyl chymotrypsin in that His-57 is protonated; however, the peptidyl group and tetrahedral carbon are absent, so that I is an imperfect analog observed only at low temperatures and pH. Much better mimics are the tetrahedral complexes of chymotrypsin with peptidyl trifluoromethylketones(II). These are excellent analogs of tetrahedral intermediates and display the LBHB at δH 18.6–18.9 ppm, which varies with the structure of the peptidyl group (24Cassidy C.S. Lin J. Frey P.A. Biochemistry. 1997; 36: 4576-4584Crossref PubMed Scopus (174) Google Scholar, 30Liang T.-C. Abeles R.H. Biochemistry. 1987; 26: 7603-7608Crossref PubMed Scopus (176) Google Scholar). The low field proton is observed at pH 4–12 and above 30 °C. The N–O distance for the LBHB is 2.5 Å when the peptidyl group isN-Ac-Leu-Phe and 2.6 Å when it is N-Ac-Phe (31Brady K. Wei A. Ringe D. Abeles R.H. Biochemistry. 1990; 29: 7600-7607Crossref PubMed Scopus (130) Google Scholar). In a tetrahedral intermediate, CF3 would be replaced by the leaving group. The LBHB is postulated to stabilize the tetrahedral intermediate and lower the activation energy for its formation (4Frey P.A. Whitt S. Tobin J. Science. 1994; 264: 1927-1930Crossref PubMed Scopus (739) Google Scholar). Stabilization by the LBHB in I and II should increase the basicity of His-57. This is observed for II, in which the apparent value of pK at 5 °C is 12.1 when the peptidyl group is N-Ac-Leu-Phe and 10.6 when it isN-Ac-Phe (24Cassidy C.S. Lin J. Frey P.A. Biochemistry. 1997; 36: 4576-4584Crossref PubMed Scopus (174) Google Scholar). The difference in pK between 12.1 and the value of 6.8 for glycyl histidine indicates ∼7.3 kcal/mol of stabilization by the LBHB (24Cassidy C.S. Lin J. Frey P.A. Biochemistry. 1997; 36: 4576-4584Crossref PubMed Scopus (174) Google Scholar). However, the apparent value of pK for His-57 in free chymotrypsin, I, is in the normal range, 7.5 at 3 °C (32Robillard G. Shulman R.G. J. Mol. Biol. 1974; 86: 541-558Crossref PubMed Scopus (94) Google Scholar). These facts can be understood by considering the differences between I and II and the properties of His-57 and Asp-102. I is a poor mimic of the transition state in that it lacks the peptidyl group and tetrahedral carbon bonded to Ser-195. Their presence leads to the increased basicity of His-57 in II. Therefore, the binding of the peptidyl group and tetrahedral carbon alters the interactions of His-57 and Asp-102 by promoting the formation of the LBHB. Conformational compression of His-57 and Asp-102 upon binding the peptidyl group and approaching the transition state would facilitate LBHB formation and increase the basicity of His-57 (24Cassidy C.S. Lin J. Frey P.A. Biochemistry. 1997; 36: 4576-4584Crossref PubMed Scopus (174) Google Scholar). Increased basicity for His-57 will enhance its reactivity as a Brønsted base in removing the proton from Ser-195 and lower the energy of the transition state for forming the tetrahedral intermediate (Fig. 2). Low field protons in trypsin, subtilisin, and α-lytic protease confirm the generality of LBHBs in serine proteases (33Markley J.L. Westler W. Biochemistry. 1996; 35: 11092-11097Crossref PubMed Scopus (94) Google Scholar, 34Halkides C.J. Wu Y.Q. Murray C.J. Biochemistry. 1996; 35: 15941-15948Crossref PubMed Scopus (72) Google Scholar). Studies of boronate complexes of chymotrypsin and other serine proteases are compatible with the compression and low barrier hydrogen-bonding mechanism. Borate, phenylboronate, and peptidylboronates form tetrahedral complexes with serine proteases through covalent bonding to Ser-195. Based on studies of the chymotrypsin-phenylboronate adduct, it was postulated that expulsion of the leaving group from the tetrahedral intermediate through general acid catalysis might be facilitated by low barrier hydrogen bonding between HNε2 of His-57 and the departing amine (35Robillard G. Shulman R.G. J. Mol. Biol. 1974; 86: 541-558Crossref PubMed Scopus (95) Google Scholar,36Steitz T.A. Shulman R.G. Annu. Rev. Biophys. Bioeng. 1982; 11: 419-444Crossref PubMed Scopus (186) Google Scholar). The importance of LBHB formation as a means of increasing the basicity of His-57 is underscored by the discovery of a class of serine proteases, such as the Escherichia coli leader peptidase, that use lysine in place of His-57 and Asp-102. The ε-amino group of lysine is a much stronger base than the imidazole ring of histidine; however, the amino group is effective only at high pH. Thus, theE. coli leader peptidase is optimally active at pH 9–10 (37Paetzel M. Strynadka N.C.J. Tschantz W.R. Casareno R. Bullinger P.R. Dalbey R.E. J. Biol. Chem. 1997; 272: 9994-10003Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). LBHB formation in chymotrypsin allows optimal activity at pH 7, with high basicity on the part of His-57, despite the fact that histidine is normally a much weaker base (pK = 6) than lysine (pK = This enzyme and a a proton from of a to the and the proton on to a The group is hydrogen-bonded to and the and show rate of and orders of magnitude, the is orders of magnitude slower than enzyme P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar). Because the pK of the intermediate in solution is J. Am. Chem. Soc. Scopus (62) Google to that for it was that a LBHB between the intermediate and the reaction and the energy for the enolization (1Gerlt J.A. Gassman P.G. Biochemistry. 1993; 32: 11934-11952Crossref PubMed Scopus (376) Google Scholar, 3Cleland W.W. Kreevoy M.M. Science. 1994; 264: 1887-1890Crossref PubMed Scopus (1062) Google Scholar). A in the A ring thus containing a in place of the at least to the than to In the neutral mimics the protonated in the intermediate complex P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar). In this proton NMR seen at and The proton at a deuterium fractionation factor of and the strength of the hydrogen bond was to be kcal/mol than one between the and water. This increase in the hydrogen bond strength corresponds to orders of magnitude rate and closely the orders of magnitude in rate in the Further has however, that the ppm proton is between and and it is the ppm proton that is between and the Biochemistry. 1997; 36: PubMed Scopus Google Scholar). the structure of the enzyme-inhibitor complex is as shown in In this structure the proton between and is from the oxygens of whereas the proton between and the intermediate or intermediate analog is bonded to the fractionation factor is Biochemistry. 1997; 36: PubMed Scopus Google Scholar)). This means that this proton is from to the substrate the enolization and thus that there is a LBHB between groups in the transition This enzyme to an was that when as a general base to a proton from the substrate, the could form a LBHB with which hydrogen bonds to the oxygen of the substrate (1Gerlt J.A. Gassman P.G. Biochemistry. 1993; 32: 11934-11952Crossref PubMed Scopus (376) Google Scholar, 3Cleland W.W. Kreevoy M.M. Science. 1994; 264: 1887-1890Crossref PubMed Scopus (1062) Google Scholar). The pK of would be 14 in solution is not protonated on the enzyme Biochemistry. PubMed Scopus Google which is close to the pK of an The close pK match and would the hydrogen bond in the initial enzyme-substrate and thus the energy would be available to the is a tightly that is to mimic the intermediate (Scheme Recent NMR studies P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar, Biochemistry. 1997; 36: PubMed Scopus Google Scholar, Biochemistry. 1997; 36: PubMed Scopus Google Scholar) have shown that the structure of the is as shown in and a LBHB is postulated between and the oxygen Biochemistry. 1997; 36: PubMed Scopus Google Scholar). The proton in this bond has a chemical shift of ppm ppm downfield from the position of the proton in acid in dimethyl and a deuterium fractionation factor of The proton of a hydrogen bond with the oxygen of the with a chemical shift of ppm ppm downfield from its position in free and a fractionation factor of is thus a strong but not low barrier hydrogen bond. The proton of the is hydrogen-bonded to with a chemical shift of and P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar, Biochemistry. 1997; 36: PubMed Scopus Google Scholar, Biochemistry. 1997; 36: PubMed Scopus Google Scholar) suggest that a role in protons to and from the oxygens of the intermediate, as well as in removing a proton from substrate, a mechanism to that postulated for the by and G.A. Biochemistry. PubMed Scopus (130) Google Scholar). However, the pKs of the and the intermediate are The LBHB between and the oxygen of the as the of a pK match between groups the oxygen and the pK of the probably to or In the intermediate, however, the pK match will be between and either oxygen of the The fractionation factor of the proton between the and is 0.7, increased hydrogen bond strength over a weak hydrogen bond. a in pK match of pK the LBHB well form with as (1Gerlt J.A. Gassman P.G. Biochemistry. 1993; 32: 11934-11952Crossref PubMed Scopus (376) Google Scholar, 3Cleland W.W. Kreevoy M.M. Science. 1994; 264: 1887-1890Crossref PubMed Scopus (1062) Google Scholar). This enzyme and of the on to which is the proton from whereas to be is hydrogen-bonded to the oxygen Struct. Biol. 2: Scopus Google Scholar). In the intermediate the close pK match between the and should a LBHB to form, thus the energy for enolization (1Gerlt J.A. Gassman P.G. Biochemistry. 1993; 32: 11934-11952Crossref PubMed Scopus (376) Google Scholar, 3Cleland W.W. Kreevoy M.M. Science. 1994; 264: 1887-1890Crossref PubMed Scopus (1062) Google Scholar). An of with or analogs of a very short hydrogen bond between the or group corresponds to the carbon of and D.P. Biochemistry. 1994; PubMed Scopus (94) Google Scholar). Although the constant of the was that of the as the pH was that the be with the proton between and the of the Because the difference in constants between the and is only a factor of 20 at pH the that the short hydrogen bonds did not in strength by more than this despite the apparent differences in degree of in the two However, the orders of magnitude than and thus the LBHB shown to have a chemical shift of 20 ppm D. D. and A. in is at least this much to As in the the LBHB with a tightly is not to the histidine, where it will be in the normal mechanism but to the In each this from the pKs of the lower than that of the intermediate and the better pK match to the group. the of the of an and are the and respectively, that the to the This reaction and the physical for stabilization of the intermediate the for the of the proposal that LBHBs are important in enzymatic catalysis (1Gerlt J.A. Gassman P.G. Biochemistry. 1993; 32: 11934-11952Crossref PubMed Scopus (376) Google Scholar, 2Gerlt J.A. Gassman P.G. J. Am. Chem. Soc. 1993; 115: 11552-11568Crossref Scopus (386) Google the pK of the of the of is whereas the pKs of the conjugate acids of the that the proton are is Thus, of the intermediate that it be The structure of a complex with that the group of the is to an and hydrogen-bonded to by the O–O J.A. J.A. G.A. Biochemistry. 1994; PubMed Scopus Google Scholar). As the substrate is converted to the enolate the on the oxygens increase to oxygen Although this increase will increase the strength of the with the the hydrogen bond strength to is also to the pK of the conjugate acid of a acid group is approximately whereas the pK of the acid is Thus, as the intermediate is the pK of the hydrogen-bonded oxygen will increase that of of the is by a factor of that the increased strength of the hydrogen bond between the enolate and relative to that the substrate at least kcal/mol to the stabilization of the intermediate A.T. J.A. G.A. Biochemistry. 1995; 34: PubMed Scopus Google Scholar). Because the that a intermediate is also in the reaction catalyzed by and a hydrogen bond is observed between and observations the proposal that increases in hydrogen bond strength are possible when the pKs of the active site and become more closely matched. In LBHBs to play a role in a number of enzymatic including the serine proteases and enzymes that their In enzymes such as use as T.M. Biochemistry. 1996; 35: PubMed Scopus Google Scholar). enzymatic reaction in which proton transfer from a general acid or to a general base an LBHB. This does not include and other factors are that LBHBs can provide at least 5 orders of magnitude in rate with other factors the of the catalysis.