NM:R Evidence for the Participation of a Low-Barrier Hydrogen Bond in The

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 8220-8224, August 1996 Biochemistry

NM:R evidence for the participation of a low-barrier hydrogen bond in the mechanism of A5-3-ketosteroid isomerase (proton transfer/catalysis/fractionation factor/ligand binding/enolization) QINJIAN ZHAOab, CHITRANANDA ABEYGUNAWARDANAa, PAUL TALALAYb, AND ALBERT S. MILDVANac Department of aBiological Chemistry and bPharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Contributed by Paul Talalay, May 15, 1996

ABSTRACT A5-3-Ketosteroid isomerase (EC 5.3.3.1) pro- the mechanism of isomerase would resolve a perplexing ques- motes an allylic rearrangement involving intramolecular pro- tion concerning the large and unfavorable difference in pKa ton transfer via a dienolic intermediate. This enzyme en- values between the 3-carbonyl group of the substrate (pKa = hances the catalytic rate by a factor of 1010. Two residues, -7) and that of the general acid, Tyr-14 (pKa = 11.6) (7). The Tyr-14, the general acid that polarizes the steroid 3-carbonyl concerted enolization is facilitated by stabilization of the group and facilitates enolization, and Asp-38 the general base incipient dienolate through unusually strong hydrogen bond- that abstracts and transfers the 4(3-proton to the 6j3-position, ing with Tyr-14. The strength of this hydrogen bond (H-bond) contribute 104.7 and 105-6 to the rate increase, respectively. A is at least 7.6 kcal/mol, as estimated from the relative k0ff major mechanistic enigma is the huge disparity between the values of the intermediate dienolate from the Y14F mutant pK, values of the catalytic groups and their targets. Upon which lacks Tyr-14 (40 s-1), and the D38N mutant which binding of an analog of the dienolate intermediate to isomer- retains Tyr-14 (s9.4 x 10-5 s-1) (5). The rate constant (koff) ase, proton NMR detects a highly deshielded resonance at cannot be measured on the wild-type enzyme because of rapid 18.15 ppm in proximity to aromatic protons, and with a 3-fold product formation. This strong H-bond has two characteristics preference for protium over deuterium (fractionation factor, of an LBHB: approximately matched pKa values of the donor 4) = 0.34), consistent with formation of a short, strong and acceptor, and exclusion of bulk solvent (6, 9). Thus, the (low-barrier) hydrogen bond to Tyr-14. The strength of this PKa of Tyr-14 in the free enzyme is 11.6 (7), whereas the pKa hydrogen bond is estimated to be at least 7.1 kcal/mol. This value of the dienolic intermediate is 10 in water (12) and is bond is relatively inaccessible to bulk solvent and is pH likely to be higher on the enzyme. The local dielectric constant insensitive. Low-barrier hydrogen bonding of Tyr-14 to the near Tyr-14 in the free enzyme is =18, as estimated by three intermediate, in conjunction with the previously demon- independent methods (7), and probably further decreases in strated tunneling contribution to the proton transfer by the enzyme-steroid complex, suggesting exclusion of solvent Asp-38, provide a plausible and quantitative explanation for from the active site. The present studies provide direct NMR the high catalytic power of this isomerase. evidence for the existence of an LBHB between Tyr-14 and the dienolic intermediate of isomerase. A5-3-Ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni is a homodimeric protein containing 125 amino MATERIALS AND METHODS acids in each subunit. This enzyme converts A5-3-ketosteroids to A4-3-ketosteroids via a dienolic intermediate with diffusion- Materials. 1713-Dihydroequilenin (1,3,5(10),6,8-estrapen- controlled efficiency. Concerted general acid-base catalysis by taene-3,17,B-diol) (DHE), estradiol 17f3-hemisuccinate, 4-flu- two amino acid residues is essential for achieving this catalytic oroestradiol, and 19-nortestosterone hemisuccinate (obtained perfection (1,2). In the rate-limiting substrate enolization step, from Steraloids, Wilton, NH) all showed a single spot on TLC Asp-38 removes the 413-proton of the steroid while Tyr-14 and were used without further purification. Ammonium sul- stabilizes the developing dienolate intermediate. In the sub- fate, buffer salts, sodium chloride, deuterium oxide, and sequent reketonization step, Asp-38 delivers the conserved perdeuterated dimethyl sulfoxide (DMSO-d6) were from proton to the 613-position, and the interaction of the steroid Sigma. Sodium 4,5-dihydroxynaphthalene-2,7-disulfonate was with Tyr-14 weakens (see Fig. 1). The origin of the large from Aldrich. catalytic rate enhancement (-1010) of the isomerase has been Sample Preparation. The D38N, Y55F/Y88F, and Y14F/ a continuing and perplexing issue (3). Here, we report direct Y88F mutant isomerase enzymes were prepared, and their NMR spectral evidence for the existence of a short, strong or concentrations were determined as reported (1, 7, 13-15). low-barrier hydrogen bond (LBHB) between the acid catalyst, NMR samples were prepared in 10 mM sodium phosphate, 20 Tyr-14, and an analog of the dienolate intermediate in the mM sodium chloride, and 9% (vol/vol) DMSO-d6 at indicated reaction mechanism of A5-3-ketosteroid isomerase. We sug- pH values. DMSO was added to the system to stabilize the gest that the LBHB is a significant contributor to the inter- isomerase in aqueous solution and to permit lowering of the mediate stabilization and catalytic power of the enzyme. temperature to below 0°C. Its presence does not significantly Nuclear Overhauser effect (NOE) studies have shown that affect the activity or structure of isomerase (14). pH values Tyr-14 closely approaches the A ring of a bound 3-ketosteroid were measured at 22°C in the presence of 9% (vol/vol) (4), and kinetic studies of the Y14F mutant indicate that DMSO-d6. Tyr-14 contributes a factor of -1047 to the total catalytic power (1010) of the isomerase (3,5). Although the existence of Abbreviations: D38N, mutant isomerase in which Asp-38 is replaced an LBHB between Tyr-14 and the dienolate intermediate on by asparagine; DHE, 173-dihydroequilenin (1,3,5(10),6,8-estrapen- the isomerase has been suspected (6-11), direct observation of taene-3,1713-diol); DMSO-d6, perdeuterated dimethyl sulfoxide; H- this LBHB has been elusive. The participation of an LBHB in bond, hydrogen bond; isomerase, A5-3-ketosteroid isomerase (EC 5.3.3.1); LBHB, low-barrier hydrogen bond; NOE, nuclear Overhauser effect; Y14F/Y88F, mutant isomerase in which Tyr-14 and Tyr-88 are The publication costs of this article were defrayed in part by page charge replaced by phenylalanine; Y55F/Y88F, mutant isomerase in which payment. This article must therefore be hereby marked "advertisement" in Tyr-55 and Tyr-88 are replaced by phenylalanine. accordance with 18 U.S.C. §1734 solely to indicate this fact. cTo whom reprint requests should be addressed. 8220 Downloaded by guest on October 1, 2021 Biochemistry: Zhao et al. Proc. Natl. Acad. Sci. USA 93 (1996) 8221 Conditions for NMR spectroscopy of enzymes and enzyme- hydroxyl proton in a random coil (21). The extraordinarily steroid complexes are given in the figure legends. The CE and strong deshielding of this proton suggests that it is located C8 proton resonance of Tyr-14 (6.78 and 6.88 ppm, respec- almost symmetrically between two oxygen atoms. tively) were assigned previously (4, 14). The spectra of ionized The Y55F/Y88F mutant of isomerase shows a kcat/Km value DHE were recorded in 0.1 M NaOH in presence of 9% of -80% of the wild-type enzyme and contains Tyr-14 as the (vol/vol) DMSO-d6 at -3.3°C. The resonances of aromatic sole tyrosine residue. When this enzyme was complexed with protons in the A and B rings of the steroid were assigned based a more soluble intermediate analog, estradiol 17,B-hemisucci- on one-dimensional proton NOEs. nate, a low field resonance (18.15 ppm) was also observed, Determination of the Fractionation Factor. The general whereas this peak was absent in the complex between this procedure of Loh and Markley (16) was used. Six samples with enzyme and 19-nortestosterone hemisuccinate (data not varying amounts of H20/D20 (12.4, 25.3, 40.3, 55.2, 70.1, and shown). Furthermore, this peak and the 11.60-ppm peak were 83.0% mole fraction H20) were prepared from 110 ,ul of a not observed under the same conditions in the absence of stock solution containing 1.5 mM D38N mutant in 10 mM Tyr-14 (i.e., with the Y14F/Y88F double mutant complexed sodium phosphate, measured pH 7.2, in D20 (4.2% H20), by with estradiol 17,B-hemisuccinate). Two further observations parallel dilutions (to 600 pl) with H20/D20 mixtures of known are consistent with an LBHB. First, replacement of DHE (pKa ratios. DHE was added to each solution (134 mM solution in = 9.0) on the D38N mutant with 4-fluoroestradiol (pKa = 7.4), DMSO-d6) to a final concentration of 0.45 mM. All solutions which increases the difference in pKa from that of Tyr-14, contained 9.0% (vol/vol) DMSO-d6. The mixtures were incu- decreases the deshielding effect, shifting the resonance from bated at 4°C for 30 min before NMR experiments. No changes 18.15 ppm to 16.41 ppm at -3.3°C. Second, this site strongly in intensities of the resonance at 18.15 ppm were seen in prefers protium to deuterium (22). Thus, a plot of the intensity representative samples after 1 week at 4°C. of the 18.15 ppm resonance as a function of the mole fraction of H20 (16) (Fig. 3) yields an equilibrium constant or fractionation factor 4 = [Enz-D][H2O]/[Enz-H][D2O] of 0.34 ± RESULTS AND DISCUSSION 0.02, consistent with a short, strong H-bond (22). To our Detection and Properties of the Low Field Resonance. knowledge, this observation represents the first spectral evi- Direct observation of the low field resonance by NMR was dence for the occurrence of an intermolecular LBHB between facilitated by use of the D38N mutant of isomerase, complexed an enzyme and a dissociable ligand. with dihydroequilenin (Fig. 1), a tightly bound analog of the NOE action spectra indicate proximity of the proton at 18.15 dienolic intermediate (12, 17, 18). The D38N mutant, which is ppm to aromatic protons with broad resonances at 6.8, 6.9, and uncharged at residue 38, thereby mimics wild-type isomerase 7.7 ppm assignable to the C_ and Ca proton resonances of when complexed with the dienolate intermediate, a complex in Tyr-14 (6.78 and 6.88 ppm, respectively) and/or to the C-4, which Asp-38 would be protonated. Accordingly, the D38N C-2, and C-1 protons of DHE (6.80, 6.96, and 7.76 ppm, mutant binds DHE at least 1000-fold more tightly than does respectively). The DHE complex with a mutant of isomerase wild-type isomerase (12). Y14F/Y88F, in which Tyr-14 was replaced by phenylalanine, In the free D38N mutant, a downfield resonance at 12.82 did not show the 18.15-ppm resonance, further supporting the ppm was observed at -3.3°C (Fig. 2A), while the free ligand assignment of this signal to a proton in an LBHB between alone, dissolved in CDCl3, gave a hydroxyl resonance at 5.59 Tyr-14 and the anion of DHE, an analog of the dienolate ppm that disappeared upon addition of a small amount of D20 intermediate. As also detected by one-dimensional proton (not shown). In the enzyme-DHE complex, three low field NOEs, proximity of the proton that resonates at 18.15 ppm to resonances were observed at 18.15, 12.89, and 11.60 ppm (Fig. an unassigned exchangeable proton that resonates at 11.60 2B). The highly deshielded signal at 18.15 ppm is in the ppm (X-H) is also absent in the mutant lacking Tyr-14 appropriate range for an LBHB (16-20 ppm) (20), represent- suggesting that the LBHB may be part of a hydrogen-bonded ing an 8.4 ppm downfield shift from that of the tyrosine network involving the dienolic intermediate as proposed (Fig. x x I - N

_W. 0 H ||| ---- Tyr-14-OH----M< Tyr-14-O06- 6- _0- 't Tyr-14-OH H* H *HAO38 H H* Asp-38 Asp-38 Asp-38

H 13' Na+

. I H O-- Y 9, 0 x+y=1 HO Dihydroequilenin (I) Sodium 4,5-dihydroxynaphthalene 2,7-disulfonate (II)

FIG. 1. Reaction mechanism of A5-3-ketosteroid isomerase, in which an LBHB contributes to the differential stabilization of the dienolic intermediate, compensated by a second H-bond donor (X-H). Structures of dihydroequilenin (DHE, I), an analog of the intermediate, and of a model compound, sodium 4,5-dihydroxynaphthalene-2,7-disulfonate (II). Downloaded by guest on October 1, 2021 8222 Biochemistry: Zhao et al. Proc. Natl. Acad. Sci. USA 93 (1996)

0.20

0.18 A

0.12 47)

a 0.08 4. B 0.04 _ , 1.0 0 2 4 6 8

0.00 I (1x 0.0 0.2 0.4 0.6 0.8 1.0 mole fraction H20 FIG. 3. Plot of peak area of the 18.15-ppm resonance normalized to the peak area of upfield nonexchangeable methyl signals (-0.07 to C -0.46 ppm) of the D38N-DHE complex as a function of the mole fraction of H20 in the aqueous solution. Nonlinear least squares fit of the data yielded a fractionation factor, 4 = [Enz-D][H20]/[Enz- H][D201 of 0.34 ± 0.02, as shown by the curve. (Inset) Linear plot (16) of the data yielded a fractionation factor of 0.330 ± 0.005, in which y is the fractional intensity of the 18.15 ppm peak in the H2O/D20 mixture compared with 100% H20, and x is the mole fraction of H2O in the solvent. the LBHB becomes accessible to solvent in only 2.5% of the D catalytic turnovers. This ratio is comparable with the 4-6% incorporation of solvent protons into the reaction product during catalysis (17, 23). The kinetic barrier to solvent proton exchange with the LB{B (AGt = 13.2 kcal/mol) is predominantly enthalpic as indicated by an activation energy of 12.9 kcal/mol and an activation enthalpy of 12.3 kcal/mol (Table 1). The exchange rate of the LBHB in isomerase is 25-fold 19-.0 18-.0 17.0 16.0 15.0 14.0 13.0 12.0 11.0 10.0 slower than the exchange rate of tyrosine hydroxyl protons in Chemical Shift, (ppm) unstructured peptides (at 4°C) (21) and is independent of pH over the range 4.3 to 9.0 (at -3.3°C). In contrast, tyrosine FIG. 2. 'H NMR observation of an LBHB (18.15 ppm) in the hydroxyl proton exchange in unstructured peptides shows both D38N-DHE complex as a function of pH. 'H NMR spectra of 0.2 mM specific-acid and specific-base catalysis (21). Our inability to free D38N mutant of isomerase at pH 7.6 (A), and 0.5 mM D38N- alter the 18.15-ppm resonance at pH values as low as 4.3, by DHE complex at pH 7.6 (B), at pH 9.0 (C), and at pH 6.6 (D). All of of DHE in = the NMR samples contained 9% (vol/vol) DMSO-d6, and pH readings protonation enzyme-bound (pKa H20 9.0), were obtained in the presence of DMSO. NMR spectra were obtained provides an independent estimate of the strength of the LBHB with a Varian Unityplus 600 MHz spectrometer at -3.3°C using the as > 7.1 kcal/mol.d 1331 pulse sequence (19) to avoid water excitation. The delays between These results indicate that the proton in the LBHB is not the pulses were adjusted for maximum excitation at 18.15 ppm, i.e. readily accessible to H30 or OH- ions and that a pH- 7800 Hz away from the carrier that is positioned at H20. Other independent conformation change of the protein, which in- parameters were a 1.5-s relaxation delay, 512-ms acquisition time, and creases its accessibility to solvent, limits the observed exchange 26-ps 90' pulse width. Spectra were processed with 10 Hz line rate. This mechanism is consistent with (i) the large enthalpic broadening, except forA, which was 40 Hz, and were zero-filled to 16 k kinetic barrier to proton exchange, (ii) the low dielectric before Fourier transformation. constant at the active site of isomerase (7), and (iii) the 1). This proposal is further supported by the upfield shifting of pH-independence of kcat over the range 6.5 to 8.5 of wild-type both peaks (18.15 and 11.60 ppm) by 1.7 and 1.0 ppm, respec- isomerase and its profoundly catalytically inefficient Y14F and tively, when DHE was replaced by 4-fluoroestradiol (data not D38N mutants (1). shown). Such a compensatory H-bond to Tyr-14 has been pre- Model Studies of an LBHB in Aqueous Solution and its viously proposed based on the UV resonance Raman and UV Exchange Behavior. In a model for the LBHB in the isomerase steroid-bound isomerase complex between a phenolic hydroxyl group and a phenolate spectra of (13, 15). with matched pKa values, we have also detected a low field Exchange Behavior of the Low Field Resonance. The signal proton resonance (17.72 ppm, 9% DMSO-d6; 17.40 ppm, 9% at 18.15 ppm is abolished by saturation of the water resonance, D20) in aqueous solution of semi-ionized sodium 4,5- indicating that this proton exchanges with water protons. The dihydroxynaphthalene-2,7-disulfonate (II). The direct obser- resonance width increases with increasing temperature, indicating a predominant exchange contribution to 1/T2. Analysis dThis lower limit value of H-bond strength is relative to that of the of the temperature dependence of 1/T2 (Fig. 4) yields an H-bond formed between DHE, a phenolic compound, and water which exchange rate of 1330 s- at 25°C, which is 40-fold slower than is -3 kcal/mol. Hence, the absolute strength of the LBHB between kcat of the wild-type enzyme (53,600 s-1) (1), suggesting that DHE and Tyr-14 is >10 kcal/mol. Downloaded by guest on October 1, 2021 Biochemistry: Zhao et al. Proc. Natl. Acad. Sci. USA 93 (1996) 8223 kcal/mol). However, the exchange mechanism differs for the model system since specific-base catalysis is detected by the 7.6 pH-dependence of the exchange rate over the limited pH range (10.0 to 12.2) that could be investigated, and by a significant entropic contribution to the kinetic barrier (-TASt = 4 7.2 kcal/mol), which is consistent with a second order process. Thus, an LBHB can exist even with some access to water, although the H-bond energy is likely to be larger in low dielectric 6.8 environments (10). Another relevant model study in tetrahydro- furan has detected a strong H-bond between 3,4-dinitrophenol and 3,4-dinitrophenolate (6.0 kcal/mol) and 4-nitrophenol and 6.4 4-nitrophenolate (6.3 kcal/mol) (ApKa = 0) (24). Mechanistic Implications. The propensity of Tyr-14 to form V- a short, strong (>7.1 kcal/mol) H-bond (most likely an LBHB) 6.0 to the incipient dienolate intermediate, in concert with the abstraction of the 4p3-proton of the substrate by Asp-38, avoids the thermodynamically and kinetically difficult task of com- 5.6 pleting the transfer of a proton from Tyr-14 (pKa = 11.6) to the 3-carbonyl group of the substrate (pKa = -7) and provides an explanation for the 104.7-fold contribution of Tyr-14 to the 5.2 catalytic power of the isomerase (1, 3). This interaction requires that Tyr-14 closely approaches the bound steroid, as observed by NOE studies (4). The similar pKa values of the 4.8 catalytic tyrosine residue (ApKa ' 1.6) and the dienolic intermediate make the formation of an LBHB highly plausible. These interpretations are not only consistent with all kinetic 3.3 3.4 3.5 3.6 3.7 3.8 and spectral evidence (10) but also explain the stereospecificity and proton conservation during the isomerization since the 1000/T91000/T,1/K intermediate would not be released from the active site until and FIG. 4t. Arrhenius plots of the effect of temperature on the the postulated LBHB is weakened by reketonization ... . ~~comc letion of the reaction. observedI transverse relaxation rates (1/T2 = X - line width) of the 18.15 Ppim peak (0). The average 1/T2 values of the two most The concerted action of Asp-38 contributes a factor of downfieliLd amides at 10.24 and 10.09 ppm (A), and the average 1/T2 105-6 to the catalytic power of the isomerase (1, 3). Here also, of six up:field methyl signals (from -0.07 to -0.46 ppm) (-) are also the source of this large factor could well be the close approach shown a,s a function of temperature. Lorentzian curve fitting of the of Asp-38 (pKa = 4.7) to the 4(3-proton of the substrate (pKa spectra vwas used to determine the line widths of these peaks. Solid = 12.7) (12), which would narrow the barrier width and straight Ilines are the theoretical lines from the linear least square fits thereby facilitate the otherwise unfavorable proton transfer. A for the 1[/T2 data of the amide and methyl proton signals. Assuming narrow barrier width is indicated by the detection of a tun- the 18.1'5-ppm resonance to have the same activation energy (-3.3 neling contribution to this proton transfer by an inflated kcal/moll) for the dipolar contribution to 1 /T2 as the average of the amide arid methyl protons, the data for the 18.15-ppm resonance were secondary kinetic isotope effect (2). Thus, both the general fitted to the Arrhenius form of the equation: 1/T2(obs) = l/T2(dipolar) acid, Tyr-14, and the general base, Asp-38, become more effective + kex, yi elding the exchange contribution (--- ). The dotted line (...) by closely approaching the substrate in a stereoelectronically is the coImputed theoretical line for 1/T2(dipolar). The curve through the favorable geometry and by acting in concert in a medium of low data is calculated for the composite results for both the dipolar and dielectric constant. Together, these effects provide a quantitative exchangee contributions to 1/T2. The probe temperature was calibrated explanation of the catalytic power of isomerase. with nea.t methanol. The contribution of strong LBHBs to the catalytic power of enzymes has been the subject of much recent discussion (6, 9, vation by NMR in aqueous solution of an OH--O- short, 20) and debate (25-29). Although strong LBHBs have been strong ]H-bond in a small molecule, with no significant steric proposed to participate in reactions catalyzed by hydrolases (6, hindranice allows the evaluation of its exchange process. Sim- 9, 20), lyases (9), a ligase (9), and several isomerases (7-10), ilar to the LBHB on isomerase, the exchange rate with the direct NMR spectral evidence for LBHBs exists only in serine model c-ompound is 1015 s-1 at 25°C with a free energy barrier proteases (16, 30-32) and aspartate aminotransferase (33). In of 13.3 kcal/mol, most of which is enthalpic (AHt = 9.3 both of these systems, the LBHBs are intramolecular. Al-

Table 1. Activation parameters, chemical shifts, and exchange rates of the exchange process of the LBHBs in A5-3-ketosteroid isomerase and in a model compound, 4,5-dihydroxynaphthalene 2,7-disulfonate (II) Activation parameter Chemical kex at Ea, AHt, /St,A -TASt at AG* at 250C, shift, ppma 25°C, s-lb kcal/molc kcal/mold cal/mol.Kd 25°C, kcal/mol kcal/mold Complex between the D38N 18.15 1330 12.9 ± 1.2 12.3 ± 1.2 -2.9 ± 0.3 0.9 ± 0.1 13.2 ± 1.3 mutant of isomerase and dihydroequilenin (I) Model compound (II) 17.72 1015 9.9 ± 0.7 9.3 ± 0.7 -13.4 ± 1.0 4.0 ± 0.3 13.3 ± 1.0 aChemical shifts are with respect to 3-(trimethylsilyl)propionate 2,2,3,3-d4 at -3.3°C in presence of 9% (vol/vol) DMSO-d6. bThe calculated exchange rates (kex) at 250C, were calculated from the theoretical line showing the exchange contribution for the enzyme complex in Fig. 4, and from an Arrhenius plot for the exchange rate as a function of temperature for the model compound. CActivation energy, Ea, was calculated from an Arrhenius plot of the temperature dependence of the transverse relaxation rate as shown in Fig. 4. dThe exchange rates and the rate of crossover, kBT/h (6.2 x 1012 s-1), were used to estimate the activation free energy. Downloaded by guest on October 1, 2021 8224 Biochemistry: Zhao et al. Proc. Natl. Acad. Sci. USA 93 (1996) though intermolecular LBHBs have been detected spectro- 10. Zhao, Q., Mildvan; A. S. & Talalay, P. (1995) Biochemistry 34, scopically between small molecules usually in organic solvents 426-434. (22, 34, 35), to our knowledge no such observation has been 11. Brooks, B. & Benisek, W. F. (1994) Biochemistry 33, 2682-2687. 12. Hawkinson, D. C., Pollack, R. M. & Ambulos, N. P., Jr. (1994) reported for an enzyme-ligand interaction in aqueous solution. Biochemistry 33, 12172-12183. The LBHB observed in the present work reconciles the 13. Austin, J. C., Zhao, Q., Jordan, T., Talalay, P., Mildvan, A. S. & paradox of the large and unfavorable differences in pKa values Spiro, T. G. (1995) Biochemistry 34, 4441-4447. between catalytic residues and their targets in isomerase. Our 14. Zhao, Q., Abeygunawardana, C. & Mildvan, A. S. (1996) Bio- findings suggest that LBHBs may contribute to the efficiencies chemistry 35, 1525-1532. of other enzymatic enolization, isomerization, and racemiza- 15. Zhao, Q., Li. Y.-K., Mildvan, A. S. & Talalay, P. (1995) Bio- tion reactions involving abstraction of aliphatic protons. The chemistry 34, 6562-6572. 16. Loh, S. N. & Markley, J. L. (1994) Biochemistry 33, 1029-1036. authors appreciate that the current debate on the potential role 17. Wang, S.-F., Kawahara, F. S. & Talalay, P. (1963) J. Bio. Chem. of LBHBs in enzyme-catalyzed reactions centers on the 238, 576-585. strength of such hydrogen bonds. Since the strength of most 18. Zeng, B., Bounds, P. L., Steiner, R. F. & Pollack, R. M. (1992) hydrogen bonds in proteins is 2 ± 1 kcal/mol, we believe that Biochemistry 31, 1521-1528. we have detected an unusually strong hydrogen bond >7.1 19. Turner, D. L. (1983) J. Magn. Reson. 54, 146-148. kcal/mold with many of the predicted properties of an LBHB. 20. Frey, P.A., Whitt, S. A., & Tobin, J. B. (1994) Science 264, 1927-1930. We are grateful to Dr. J. L. Markley for expert advice on measure- 21. Liepinsch, E., Otting, G. & Wuthrich, K. (1992) J. Biomol. NMR ment of the fractionation factor and discussion of his work prior to 2, 447-465. publication, to Dr. P. A. Frey for valuable comments, and to Gale 22. Kreevoy, M. M. & Liang, T. M. (1980) J. Am. Chem. Soc. 102, Doremus for in of the This work was 3315-3322. help preparation manuscript. 23. Hawkinson, D. C., Eames, T. C. M. & Pollack, R. M. (1991) supported by National Institutes of Health Grants DK 07422 (to P.T.) Biochemistry 30, 6956-6964. and DK 28616 (to A.S.M.). 24. Shan, S. O., Loh, S. & Herschlag, D. 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