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J Biol Chem, Vol. 273, Issue 40, 25529-25532, October 2, 1998
§,, and
From the Institute for Enzyme Research, University of
Wisconsin, Madison, Wisconsin 53705 and ¶ Department of
Biochemistry, University of Illinois, Urbana, Illinois 61801
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.
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 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
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 left-hand or right-hand oxygen and is more
loosely bonded to the other one, with an O-O distance of
INTRODUCTION
Top
Introduction
References
Properties of Hydrogen Bonds
H 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.
H of formation can approach 25 or 30 kcal
mol1 (16, 17). 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 (18, 19). In organic solvents, strong
hydrogen bonds can also form, although the H of formation
probably never exceeds 20 kcal mol1. 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.
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.
View larger version (6K):
[in a new window]
Fig. 1.
Energy diagrams for hydrogen bonds between
groups of equal pK. A, weak hydrogen bond
with O-O distance of 2.8 Å; the two positions of the hydrogen are
shown. B, low barrier hydrogen bond of length 2.55 Å; the
hydrogen is diffusely distributed, with average position in the center.
C, single-well hydrogen bond with length 2.29 Å. The
upper and lower horizontal
lines are zero point energy levels for hydrogen and
deuterium, and the curves define the energetic barrier to
changes in bond length. The distances are to scale.
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. 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 diffusely distributed with its
average position in the center (20). LBHBs are largely covalent (21).
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 (20). 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 between p-nitrophenol and p-nitrophenolate ion in organic solvents (22). 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. 23.
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The Role of LBHBs in Enzymatic Catalysis |
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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 (1-4). 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 (4, 24).
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 pK
values 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 (8, 25). However,
the data of Shan and Herschlag (25, 26) show that the variation in
G of formation with pK depends on the
solvent. Although the slope of a plot of the log of the formation
constant (Kform) versus
pK 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 pK
values. If this change pertains over the full range of pK
values, a change of 5 to 14 in pK would provide over 18 kcal mol1 of energy, which corresponds to over 13 orders
of magnitude in rate acceleration. Although the slope of
log(Kform) versus pK probably does not remain constant as the pK increases, it
is clear that considerable energy is potentially available to help catalyze enzymatic reactions.
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Acid-Base Catalysis |
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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
(27)).
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Serine Proteases |
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A clear example of a LBHB in enzymatic catalysis is shown by
chymotrypsin. The low field proton seen at 
18.3 ppm for the proton
between His-57 and Asp-102 in acidic solutions of chymotrypsin (28) has
been assigned as a LBHB (4). Chemical models in nonaqueous media
confirm the low field NMR signal, as well as reveal characteristic
Fourier transfer IR features (29). The LBHB-containing structure
I (Scheme I) displays the low field proton signal as well as a very short N-O distance of <2.6 Å between the heteroatoms. The van der Waals contact distance for
nitrogen and oxygen is 2.7 Å. The LBHB partially depolarizes the ion
pair between His-57 and Asp-102, so that in structures I and
II (Scheme I), 1 > 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 H 18.6-18.9 ppm,
which varies with the structure of the peptidyl group (24, 30). 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 is
N-Ac-Leu-Phe and 2.6 Å when it is N-Ac-Phe (31).
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 (4).
|
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 is
N-Ac-Phe (24). 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 (24). However, the apparent value of
pK for His-57 in free chymotrypsin, I, is in the
normal range, 7.5 at 3 °C (32). 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 (24). 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 
-lytic protease confirm the generality of LBHBs in
serine proteases (33, 34).
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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 HN
2 of His-57 and the departing amine (35,
36).
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 
-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, the E. coli leader peptidase is optimally active at pH 9-10
(37). 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 = 10.5).
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Ketosteroid Isomerase |
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This enzyme interconverts non-conjugated and conjugated
ketosteroids via a dienolate intermediate. Asp-38 removes a proton from
C-4 of a 5 ketosteroid to give the dienolate and puts
the proton back on C-6 to give a 4 product. The keto
group is hydrogen-bonded to Tyr-14, and the Y14F and D38N isomerases
show rate reductions of 4.7 and 5.6 orders of magnitude, respectively;
the double mutant is ~10 orders of magnitude slower than wild type
enzyme (38). Because the pK of the dienol intermediate in
solution is 10 (39), (close to that for tyrosine), it was proposed that
a LBHB formed between the intermediate and Tyr-14 during the reaction
and provided the energy for the enolization (1, 3).
A steroid aromatic in the A ring (and thus containing a phenolic hydroxyl in place of the ketone) bound at least 1000-fold tighter to the D38N varient than to wild type isomerase. In D38N the neutral Asn-38 mimics the protonated Asp-38 in the intermediate enzyme-dienolate complex (38). In this complex, proton NMR peaks were seen at 18.15 and 11.6 ppm. The proton at 18.15 displayed a deuterium fractionation factor of 0.34, and the strength of the hydrogen bond was estimated to be 7.1 kcal/mol greater than one between the inhibitor and water. This increase in the hydrogen bond strength corresponds to 5.2 orders of magnitude rate acceleration and closely matches the 4.7 orders of magnitude decrease in rate in the Y14F mutant.
Further work has shown, however, that the 18.15 ppm proton is actually between Tyr-14 and Asp-99, and it is the 11.6 ppm proton that is between Tyr-14 and the phenolic inhibitor (40). Thus the structure of the enzyme-inhibitor complex is as shown in Scheme II. In this structure the proton between Asp-99 and Tyr-14 is presumably equidistant from the oxygens of both groups, whereas the proton between Tyr-14 and the intermediate or intermediate analog is mainly bonded to the latter (its fractionation factor is 0.97 (40)). This means that this proton is transferred from Tyr-14 to the substrate during the enolization and thus that there presumably is a LBHB between these groups in the transition state.
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Triose-phosphate Isomerase |
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This enzyme isomerizes dihydroxyacetone phosphate to glyceraldehyde 3-phosphate via an endiol(ate) intermediate. It was proposed that when Glu-165 acted as a general base to remove a proton from the substrate, the resulting enediolate could form a LBHB with His-95, which hydrogen bonds to the carbonyl oxygen of the substrate (1, 3). The pK of His-95 would be 14 in solution (it is not protonated on the enzyme (41)), which is close to the expected pK of an enediol. The close pK match and ideal geometry would strengthen the hydrogen bond (weak in the initial enzyme-substrate complex), and thus the liberated energy would be available to speed up the enolization.
Phosphoglycolohydroxamate is a tightly bound inhibitor that is thought
to mimic the enediol(ate) intermediate (Scheme
III). Recent NMR studies (38, 40, 42)
have shown that the actual structure of the bound hydroxamate is as
shown in Scheme IV, and a LBHB is
postulated between Glu-165 and the hydroxamate oxygen (42). The proton
in this bond has a chemical shift of 14.9 ppm (6 ppm downfield from the
position of the analogous proton in acetohydroxamic acid in dimethyl
sulfoxide) and a deuterium fractionation factor of 0.38. The N-
proton of His-95 forms a hydrogen bond with the carbonyl oxygen of the
inhibitor with a chemical shift of 13.5 ppm (0.4 ppm downfield from its
position in free enzyme) and a fractionation factor of 0.71. It is thus
a strong but not low barrier hydrogen bond. The NH proton of the
inhibitor is hydrogen-bonded to Glu-165 with a chemical shift of 10.9 ppm.
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Mildvan and co-workers (38, 40, 42) suggest that Glu-165 plays a role in moving protons to and from the oxygens of the intermediate, as well as in initially removing a proton from substrate, a mechanism similar to that postulated for the H95Q mutant by Knowles and co-workers (43). However, the pKs of the hydroxamate (9.5) and the enediol intermediate (~14) are quite different. The LBHB forms between Glu-165 and the hydroxamate oxygen of the inhibitor as the result of a favorable pK match between these groups after His-95 polarizes the carbonyl oxygen and lowers the pK of the inhibitor, probably to 7 or below. In the enediol intermediate, however, the best pK match will be between His-95 and either oxygen of the enediol. The fractionation factor of the proton between the inhibitor and His-95 is 0.7, showing increased hydrogen bond strength over a weak hydrogen bond. With a further improvement in pK match of perhaps 7 pK units, the LBHB may well form with His-95, as originally proposed (1, 3).
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Citrate Synthase |
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This enzyme enolizes acetyl-CoA and catalyzes attack of the enol(ate) on oxalacetate to give citryl-CoA, which is then hydrolyzed. Asp-375 removes the proton from acetyl-CoA, whereas His-274 (presumed to be neutral) is hydrogen-bonded to the carbonyl oxygen (44). In the enolized intermediate the close pK match between the enol and His-274 should permit a LBHB to form, thus producing the energy for enolization (1, 3). An x-ray study of citrate synthase with bound carboxyl or amide analogs of acetyl-CoA showed a very short (2.4-2.5 Å) hydrogen bond between the carboxyl or amide group (which corresponds to the methyl carbon of acetyl-CoA) and Asp-375 (45). Although the dissociation constant of the amide was pH-independent, that of the carboxyl inhibitor decreased as the pH was lowered, showing that the carboxyl must be protonated, with the proton presumably between Asp-375 and the carboxyl of the inhibitor. Because the difference in dissociation constants between the carboxyl and amide inhibitors is only a factor of 20 at pH 6, the authors concluded that the short hydrogen bonds did not differ in strength by more than this factor, despite the apparent differences in degree of pK mismatch in the two cases. However, the carboxyl inhibitor binds 4 orders of magnitude tighter than acetyl-CoA, and thus the LBHB (now shown to have a chemical shift of 20 ppm (46)) is contributing at least this much to binding.
As in the triose-phosphate isomerase case, the LBHB formed with a tightly bound inhibitor is not to the histidine, where it presumably will be in the normal catalytic mechanism but to the carboxylate base. In each case this results from the pKs of the inhibitor being lower than that of the putative intermediate and the better pK match to the carboxyl group.
| |
Mandelate Racemase |
|---|
Mandelate racemase catalyzes the interconversion of the
enantiomers of mandelate via an enol(ate) intermediate. Lys-166 and His-297 are the (S)- and (R)-specific bases,
respectively, that abstract the -proton to generate the
intermediate. This reaction and understanding the physical basis for
stabilization of the intermediate provided the basis for the early
statements of the proposal that LBHBs are important in enzymatic
catalysis (1, 2); the pK of the -proton of the
enantiomers of mandelate is 29, whereas the pKs of the
conjugate acids of the bases that receive the proton are ~6, yet
kcat is ~500 s1. Thus, kinetic
competence of the intermediate requires that it be
stabilized. The x-ray structure of a complex with
(S)-atrolactate ((S)--methylmandelate) reveals
that the carboxylate group of the bound inhibitor is both coordinated
to an essential Mg2+ and hydrogen-bonded to Glu-317 (as
evidenced by the 2.7-Å O-O distance) (47).
As the substrate is converted to the enolate anion, the charge
densities on the carboxylate oxygens increase (from 0.5 to 1 per
oxygen atom). Although this increase undoubtedly will increase the
strength of the electrostatic interaction with the Mg2+,
the hydrogen bond strength to Glu-317 is also expected to increase; the
pK of the conjugate acid of a carboxylic acid group (or
charge-neutralized carboxylate anion) is approximately 8, whereas the
pK of the mandelic acid enol is ~7. Thus, as the enol(ate)
intermediate is formed, the pK of the hydrogen-bonded
carboxylate oxygen will increase toward that of Glu-317. The
kcat of the E317Q racemase is decreased by a
factor of ~105, suggesting that the increased strength of
the hydrogen bond between the enolate anion and Glu-317 relative to
that involving the bound substrate contributes at least 7 kcal/mol to
the stabilization of the intermediate (48). Because the kinetic
evidence suggests that a transiently stable intermediate is also formed
in the reaction catalyzed by E317Q and a hydrogen bond is observed
between Gln-317 and (S)-atrolactate, these observations
support the proposal that significant increases in hydrogen bond
strength are possible when the pKs of the active site donors
and substrate/intermediate acceptors become more closely
matched.
In conclusion, LBHBs appear to play a role in a number of enzymatic reactions, including the serine proteases and certain enzymes that enolize their substrates. In contrast, enzymes such as enolase use metal ions as electrostatic catalysts (49). Any enzymatic reaction in which proton transfer from a general acid or to a general base occurs likely may involve an LBHB. This does not include all enzymes, and other catalytic factors are clearly involved. We believe that LBHBs can provide at least 5 orders of magnitude in rate acceleration, with other factors providing the rest of the catalysis.
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FOOTNOTES |
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* This minireview will be reprinted in the 1998 Minireview Compendium, which will be available in December, 1998. This is the first article of three in the "Enzyme Catalytic Power Minireview Series." This work was supported by National Institutes of Health Grants GM-51806 (to W. W. C.), DK-28607 (to P. A. F.), and GM-40570 and GM-52594 (to J. A. G.).
§ To whom correspondence should be addressed. Tel.: 608-262-1373; Fax: 608-265-2904; E-mail: Cleland{at}enzyme.wisc.edu.
The abbreviation used is: LBHB, low barrier hydrogen bond.
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REFERENCES |
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Top
Introduction
References
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H.-S. Cho, N.-C. Ha, G. Choi, H.-J. Kim, D. Lee, K. S. Oh, K. S. Kim, W. Lee, K. Y. Choi, and B.-H. Oh Crystal Structure of Delta 5-3-Ketosteroid Isomerase from Pseudomonas testosteroni in Complex with Equilenin Settles the Correct Hydrogen Bonding Scheme for Transition State Stabilization J. Biol. Chem., November 12, 1999; 274(46): 32863 - 32868. [Abstract] [Full Text] [PDF]
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A. M. Mulichak, M. J. Theisen, B. Essigmann, C. Benning, and R. M. Garavito Crystal structure of SQD1, an enzyme involved in the biosynthesis of the plant sulfolipid headgroup donor UDP-sulfoquinovose PNAS, November 9, 1999; 96(23): 13097 - 13102. [Abstract] [Full Text] [PDF]
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A. Warshel Electrostatic Origin of the Catalytic Power of Enzymes and the Role of Preorganized Active Sites J. Biol. Chem., October 16, 1998; 273(42): 27035 - 27038. [Full Text] [PDF]
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W. R. Cannon and S. J. Benkovic Solvation, Reorganization Energy, and Biological Catalysis J. Biol. Chem., October 9, 1998; 273(41): 26257 - 26260. [Full Text] [PDF]
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N.-C. Ha, M.-S. Kim, W. Lee, K. Y. Choi, and B.-H. Oh Detection of Large pKa Perturbations of an Inhibitor and a Catalytic Group at an Enzyme Active Site, a Mechanistic Basis for Catalytic Power of Many Enzymes J. Biol. Chem., December 22, 2000; 275(52): 41100 - 41106. [Abstract] [Full Text] [PDF]
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P. Heikinheimo, V. Tuominen, A.-K. Ahonen, A. Teplyakov, B. S. Cooperman, A. A. Baykov, R. Lahti, and A. Goldman Toward a quantum-mechanical description of metal-assisted phosphoryl transfer in pyrophosphatase PNAS, March 13, 2001; 98(6): 3121 - 3126. [Abstract] [Full Text] [PDF]
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