key: cord-0747340-60rfq6kk
authors: Cleary, Jennifer A.; Doherty, William; Evans, Paul; Malthouse, J.Paul G.
title: Hemiacetal stabilization in a chymotrypsin inhibitor complex and the reactivity of the hydroxyl group of the catalytic serine residue of chymotrypsin
date: 2014-06-30
journal: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
DOI: 10.1016/j.bbapap.2014.03.008
sha: 2b82a2281c12519b59609a013a6bed2583aa8ecc
doc_id: 747340
cord_uid: 60rfq6kk

Abstract The aldehyde inhibitor Z-Ala-Ala-Phe-CHO has been synthesized and shown by 13C-NMR to react with the active site serine hydroxyl group of alpha-chymotrypsin to form two diastereomeric hemiacetals. For both hemiacetals oxyanion formation occurs with a pKa value of ~7 showing that chymotrypsin reduces the oxyanion pKa values by ~5.6 pKa units and stabilizes the oxyanions of both diastereoisomers by ~32kJmol−1. As pH has only a small effect on binding we conclude that oxyanion formation does not have a significant effect on binding the aldehyde inhibitor. By comparing the binding of Z-Ala-Ala-Phe-CHO with that of Z-Ala-Ala-Phe-H we estimate that the aldehyde group increases binding ~100 fold. At pH7.2 the effective molarity of the active site serine hydroxy group is ~6000 which is ~7× less effective than with the corresponding glyoxal inhibitor. Using 1H-NMR we have shown that at both 4 and 25°C the histidine pKa is ~7.3 in free chymotrypsin and it is raised to ~8 when Z-Ala-Ala-Phe-CHO is bound. We conclude that oxyanion formation only has a minor role in raising the histidine pKa and that the aldehyde hydrogen must be replaced by a larger group to raise the histidine pKa >10 and give stereospecific formation of tetrahedral intermediates. The results show that a large increase in the pKa of the active site histidine is not needed for the active site serine hydroxyl group to have an effective molarity of 6000.

Specific peptide aldehyde inhibitors are usually formed by replacing the carboxy terminal carboxylate group with an aldehyde group. Such substrate derived aldehyde inhibitors are often highly specific and potent inhibitors of the serine proteases. Consequently they have been used to target specific serine proteases such as prostate specific antigen [1] , the proteasome [2] and the SARS 3Cl protease [3] in the hope that they can be used to help in the treatment of conditions such as prostate cancer and severe acute respiratory syndrome.

We have recently developed an approach which enables us to quantify how much a peptide warhead contributes to inhibitor potency [4] . This has been used to determine how the glyoxylate group and the carboxylate group contribute to inhibitor potency with the serine protease chymotrypsin. In this paper we use the same approach to determine how the aldehyde group contributes to inhibitor potency with chymotrypsin.

The rate-limiting step in peptide catalysis by the serine proteases is thought to be either formation or breakdown of a tetrahedral intermediate formed by the addition of the hydroxyl group of the catalytic serine to the carbonyl carbon of the peptide bond being hydrolysed. The tight binding of aldehydes to the cysteine protease papain was attributed to the formation of a thiohemiacetal by the addition of the thiolate ion of the catalytic cysteine to the carbonyl of the peptide bond being hydrolysed analogous to the tetrahedral intermediate formed during catalysis [5] . Similar proposals were made when aldehydes were found to be potent inhibitors of the serine protease elastase [6] . Using 1 H-NMR indirect evidence for hemiacetal formation with the serine protease chymotrypsin was obtained using saturation transfer [7] [8] [9] . Direct observation of a hemiacetal signal was reported using 19 F-NMR with N-Acetyl-p-fluorophenylalinals and α-chymotrypsin [10, 11] . 13 C-NMR studies of the aldehyde inhibitor Acetyl-Phe-CHO binding to αchymotrypsin have shown two hemiacetals are formed [12] . 13 C-NMR has shown that the aldehyde inhibitor leupeptin (N-Acetyl-Leu-Leu-Leu-Arg-CHO) also forms two diastereomeric hemiacetals with trypsin [13] . However, with Acetyl-Leu-Phe-CHO it was reported that only one hemiacetal signal was observed [14] . The fact that only small changes (≤4 fold) in K i values have been observed from pH 3-8 when aldehyde inhibitors bind to chymotrypsin, has led to the suggestion that the hemiacetal formed with chymotrypsin remains neutral from pH 3-8 [11, 15] . Likewise, the fact that the chemical shifts of the hemiacetals observed with Acetyl-Phe-CHO [12] or Acetyl-Leu-Phe-CHO [14] did not titrate significantly at pHs N 7 has been used to support the suggestion that the hemiacetals are neutral from pH 3-8. However, with trypsin the hydroxy groups of both the diastereomeric hemiacetals formed with leupeptin and Acetyl-Leu-Phe-CHO ionized showing oxyanion formation with pKa values of 4.69 and 5.67 [13] . We have also shown that there is oxyanion formation (pKa~5) in chymotrypsin glyoxal inhibitor complexes formed with Z-Ala-Ala-Phe-COCHO (Scheme 1D) [16] . Therefore in this study we use 13 C-NMR to determine whether there is oxyanion formation in the corresponding chymotrypsin-aldehyde inhibitor complexes formed with Z-Ala-Ala-Phe-CHO (Scheme 1C) from pH 3 to 11. This allows us to compare how chymotrypsin stabilizes oxyanion formation in hemiacetals and hemiketals [16, 17] .

Recently we have developed a procedure that allows us to quantify hemiketal formation in chymotrypsin-glyoxal inhibitor complexes and to determine the effective molarity of the hydroxyl group of the catalytic serine residue in these complexes [4] . In this study we use a similar approach to quantify hemiacetal formation in a chymotrypsinaldehyde inhibitor complex and to determine the effective molarity of the hydroxyl group of the catalytic serine residue in this complex. We also undertake an extensive 1 H-NMR study of how the ionization state of the active site histidine residue changes when aldehyde inhibitors are bound. This allows us to determine how oxyanion formation affects the histidine pK a and also whether a high histidine pK a is required for the active site serine hydroxyl group to have a high effective molarity. Finally we discuss the mechanistic significance of these results. 

Z-Ala-Ala-Phe-H (Scheme 1A) was synthesized as described by Petrillo et al. [4] . Z-Ala-Ala-Phe-13 COOCH 3 and Z-Ala-Ala-Phe-13 COCHO (Scheme 1D) were synthesized as described by Cosgrove et al., [18] .

Z-Ala-Ala-Phe-13 COOCH 3 (220 mg, 0.48 mmol, 1 equiv.) was suspended in anhydrous tetrahydrofuran (2 mL) and added to a stirred suspension of LiAlH 4 (125 mg, 3.29 mmol, 7 equiv.) in anhydrous THF (3.5 mL) under nitrogen at 0°C. The reaction mixture was left to stir for 1 h at 0°C followed by a further 30 min at room temperature. The reaction mixture was cooled to 0°C and ethyl acetate (5 mL) was cautiously added dropwise to the mixture, followed by dropwise addition of ice cold water (5 mL). The resulting mixture was stirred for a further 30 min and the layers partitioned. The aqueous layer was extracted with ethyl acetate (2 × 10 mL) and the combined organic layers were washed with brine (10 mL), dried over MgSO 4 , filtered and solvent was removed in vacuo to give the crude product. Purification by silica chromatography using CH 2 Cl 2 -MeOH; 15:1 as an eluant gave the product as a white solid. M.p. 170-175°C.

The yield was 0.112 g (0.248 mmol 54%). 13 C-NMR analysis gave the following data. δ C (100 MHz, d 6 -DMSO) 18 

A sonicated suspension of Z-Ala-Ala-Phe-13 CH 2 OH (94 mg, 0.22 mmol, 1 equiv.) in anhydrous CH 2 Cl 2 (3 mL) was added to a stirred solution of pyridinium chlorochromate (315 mg, 1.46 mmol, 6.5 equiv.) and Celite (280 mg) in anhydrous CH 2 Cl 2 (2 mL). The reaction mixture was left to stir for 6 h after which time silica (ca. 2 g) was added and solvent was removed in vacuo. The residue was purified by silica chromatography using ethylacetate as an eluant. This gave the product as a white solid.

The yield was 0.038 g (0.085 mmol, 41%). 13 C-NMR analysis gave the following data. δ C (100 MHz, d 6 

α-Chymotrypsin (crystallized and lyophilized) was obtained from the Sigma Chemical Co. The amount of fully active enzyme (~77%) was determined as described by Finucane et al. [19] .

The K i value for inhibition of the chymotrypsin-catalyzed hydrolysis of succinyl-Ala-Ala-Pro-Phe-pna at pH 7.2 in 0.1 M potassium phosphate buffer was determined by determining K M(obs) values at different inhibitor concentrations and then plotting K M(obs) versus the inhibitor concentration. K i was determined from the intercept on the x-axis. All other K i values were determined when [S 0 ] ≪ K M by as described by [4] .

NMR spectra at 11.75 T were recorded with a Bruker Avance DRX 500 standard-bore spectrometer operating at 125.7716 MHz for 13 Cnuclei. 10 mm-diameter NMR tubes containing~3 mL samples were used for 13 C-NMR spectroscopy. The 13 C-NMR spectral conditions for the samples were: 8192 time domain data points; spectral width 236.6 ppm; acquisition time 0.138 s; 0.6 s relaxation delay time; 90°C pulse angle; 2048 transients recorded per spectrum. Waltz-16 composite pulse 1 H decoupling of 1.0 W was used which was reduced to 0.026 W during the relaxation delay to minimize dielectric heating but maintain the Nuclear Overhauser Effect. 1 H-NMR spectra were recorded at 500 MHz using 5-mm-diameter NMR tubes. Spectral conditions used for observing H-bonded protons at 12-18 ppm were: 32768 time domain data points, spectral width 40 ppm, acquisition time 0.819 s, 1.0 s relaxation delay, 90°pulse angle, 4 dummy scans and 512 transients were recorded per spectrum. Water suppression was carried out with the Watergate W5 pulse sequence [20] and the 1331 pulse sequence [21] .

All spectra were transformed using an exponential weight factor of 50 Hz. Both 1 H and 13 C chemical shifts are quoted relative to tetramethylsilane at 0.00 ppm. The chemical shift of d 6 -dimethyl sulfoxide at 38.7 ppm was used as a secondary reference for 13 C-NMR spectra obtained in aqueous solutions. Aqueous samples contained 10% (v/v) 2 H 2 O to obtain a deuterium lock signal.

In an aqueous solvent at pH 7.2 containing 10 mM sodium phosphate buffer, 10% d 6 -DMSO and 10% 2 H 2 O Z-Ala-Ala-Phe-13 CHO (Scheme 1C) gave signals at 202.7 and 91.0 ppm with J CH values of 183.0 ± 0.3 Hz and 163.4 ± 0.1 Hz respectively. T 1 (spin lattice relaxation times) were determined by the inversion recovery method. The signal at 91 ppm had a spin lattice relaxation time of 0.61 ± 0.10 s. Using quantitative 13 C-NMR it was determined that the hydration constant (K H1obs ) was 13.9 ± 0.7 (Mean of 13 determinations.).

On adding Z-Ala-Ala-Phe-13 CHO (Fig. 1a) to α-chymotrypsin ( Fig. 1b) new signals were observed at 99.2 ppm (J CH 158.8 Hz) and 95.5 ppm (J CH 169.3 Hz) at pH 2.93 (Fig. 1c) . The signal at 38.7 ppm is due the d 6 = DMSO (1.8% (v/v)) used to make the inhibitor stock solution and the signal at 91.2 ppm (J CH 163.4 Hz) (Fig. 1c ) is due to a small excess of the inhibitor (Fig. 1a) . The signals at 99.2 and 95.5 had T 1 values of 0.64 ± 0.07 s and 0.69 ± 0.08 s respectively and are assigned to diastereomeric hemiacetals formed when the active site serine hydroxyl group reacts with the 13 C-enriched inhibitor aldehyde group. A hydrate cannot form diastereoisomers and would only give one NMR signal. Therefore the fact that we observe two 13 C-NMR signals confirms that two diastereomeric hemiacetals have been formed and that the inhibitor is not bound as a hydrate. The inhibitor was tightly bound at both acid and alkaline pHs (Table 5 ) and we have calculated that more than 98% of the enzyme is saturated with inhibitor from pH 3.2 to 10.6.

Hemiacetals like hemiketals are expected to have chemical shifts 4 ppm larger than their hydrates [16] . Therefore the signal at 95.5 ppm has a chemical shift close to that expected if the hydroxyl group of the active site serine of chymotrypsin forms a hemiacetal with the inhibitor aldehyde group. If this process is stereospecific then only one hemiacetal signal will be observed. However, as two hemiacetal signals are observed then we can conclude that hemiacetal formation is not stereospecific.

From the areas of signals at 99.2 ppm and 95.5 ppm (Fig. 1c ) the proportions of the corresponding hemiacetals at pH 2.93 were 22.3 ± 0.6% and 77.7 ± 0.6% respectively. Similar proportions (22.6 ± 2.1% and 77.4 ± 2.1%) were obtained at pH 5.85 (Fig. 1e ). Due to line broadening accurate measurements could not be made at intermediate pHs. However, similar proportions were also obtained at pHs 10.26 (Fig. 1i , 20.8 ± 1.0% and 79.2 ± 1.0%) and pH 11.10 ( Fig. 1j , 26.5 ± 3.0% and 73.5 ± 3.0%). The mean values are 23 ± 2.4% for the diastereoisomer with a [16] signal at 99.2 ppm and 77 ± 2.4% for the diastereoisomer with a signal at 95.5 ppm. Using 13 C-NMR two diastereomeric thiohemiacetals were first observed when the cysteine protease papain was incubated with an aldehyde inhibitor [23] . Subsequently two hemiacetals were observed by 13 C-NMR when aldehyde inhibitors reacted with the serine proteases chymotrypsin [12] and trypsin [13] . The hemiacetals formed when leupeptin (N-acetyl-Leu-Leu-Arg-CHO) reacted with trypsin [13] had similar chemical shifts and proportions except the signal at higher chemical shift predominated (86%) whereas in the present work the signal at lower chemical shift predominated (77.4-79.2%).

The K i value for the inhibition of the chymotrypsin catalyzed hydrolysis of succinyl-Ala-Ala-Pro-Phe-pna by the aldehyde inhibitor Z-Ala-Ala-Phe-CHO at pH 7.2 was determined from the dependence of the observed K M value for succinyl-Ala-Ala-Pro-Phe-pna on the concentration of the inhibitor present. From the K i values of ZAAF-H and ZAAF-CHO we estimate that the aldehyde group increases binding~100 fold ( Table 1) .

The glyoxal group of ZAAF-COCHO is bound 4.6 times more tightly than the aldehyde group of ZAAF-CHO (Table 1 ). 13 C-NMR has shown that ZAAF-CHO reacts with chymotrypsin to form two hemiacetals, one smaller signal with a chemical shift of 99.2 ppm and the other larger signal with a chemical shift of 95.5 ppm at pH 2.93 (Fig. 1c) 

Assuming K i(obs) for ZAAF-H (Scheme 1A) was a good approximation for the disassociation constant (K s ) of the corresponding chymotrypsin-ZAAF-CHO complex (EA ald in Scheme 2) K HA(obs) (Eq. (4)) was calculated. The K HA(obs) of 1500 for hemiacetal formation with ZAAF-CHO was similar in magnitude to K HK(obs) for hemiketal formation with ZAAF-COCHO (Table 2 ). Using Eq. (3), the value of 1500 for K HK(obs) and the mean proportions of 77% and 23% for the diastereoisomers at 95.5 ppm and 99.2 ppm respectively K HA1 and K HA2 were determined for the two diastereoisomers ( Table 2) .

Hemiacetal formation in the chymotrypsin-aldehyde inhibitor complex is a first order process (K HAobs = [hemiacetal] / [EA]) whereas the hydration of the free aldehyde inhibitor in water is a second order process (K H1 = [hemiacetal] / [aldehyde][H 2 O]). K H1 is calculated by dividing K H1(obs) (K H1(obs) = [hemiacetal] / [aldehyde]) by the molarity of water. The ratio K HAobs /K H1 is therefore the effective molarity of the serine hydroxyl group in hemiacetal formation. This effective molarity is the molarity of water required to be as effective as the catalytic serine hydroxyl group in forming a hemiacetal with the inhibitor aldehyde group. The effective molarity of the active site serine hydroxyl group is 7 times lower for hemiacetal formation with ZAAF-CHO (Scheme 1C) compared to hemiketal formation with the keto carbon of the glyoxal group of ZAAF-COCHO (Scheme 1D).

3.5. Effect of pH on the 13 C-NMR signals from Z-Ala-Ala-Phe-CHO in the presence and absence of α-chymotrypsin On increasing the pH the chemical shift of both the large signal at 95.5 and the small signal at 99.2 ppm increased (Fig. 1a-j) . The chemical shift of the large signal (Fig. 1c) increased from at 95.55 ± 0.02 ppm to 97.66 ± 0.02 ppm with a pK a of 6.86 ± 0.05 (Fig. 2B) . There was also a further small increase in chemical shift to 98.99 ± 0.04 ppm with a pK a Scheme 2. Minimal scheme for hemiacetal formation by chymotrypsin. Table 2 The effective molarity of the catalytic serine of the α-chymotrypsin when forming hemiketals with glyoxal inhibitors or hemiacetals with aldehyde inhibitors at pH 7.2. of 10.65 ± 0.12 (Fig. 2B) . The total titration shift of 3.44 ppm is similar to that of 3.53 ppm observed for formation of the hemiketal oxyanion with the corresponding hemiketal formed by the glyoxal inhibitor Z-Ala-Ala-Phe- 13 COCHO reacting with chymotrypsin (Table 3) . Therefore the titration shift is assigned to formation of the hemiacetal oxyanion when Z-Ala-Ala-Phe-CHO forms a hemiacetal with chymotrypsin. Ionization of the isoleucine-16/aspartate-194 ion pair causes a conformational change which inactivates chymotrypsin [19, 24] . The binding of ligands and or chemical modification of the active site serine hydroxyl is known to raise the pKa of the isoleucine-16/aspartate-194 ion pair to a value of 10.5-11.0 [16, 19] and so we assign the pKa of 10.65 to ionization of the isoleucine-16/aspartate-194 ion pair. The smaller signal at 99.2 ppm (Fig. 1c) titrated with a similar pKa of 7.0( Fig. 2C ) but the titration shift (δ 2 -δ 1 ) was smaller (Table 3 ).

The pK a values of~7.0 for the hemiacetal oxyanions in the inhibitor complexes are much smaller than the oxyanion pK a of 13.23 ± 0.1 for the inhibitor hydrate ( Fig. 2A) . This shows that chymotrypsin is stabilizing the hemiacetal oxyanion of both diastereoisomers by similar amounts. In order to quantify this stabilization we need to determine the hemiacetal pK a in the absence of enzyme stabilization. This can be done using free energy relationships [25] . The pK a of ZAAF-13 COOH (Scheme 1B) was determined by 13 C-NMR to be 3.52 ± 0.03. Using the relationship σ* = (4.644-pKa)/1.7 [26] for ZAAF-COOH the substituent constant, σ* = 0.66 for the substituent ZAAF\. The substituent constant for EO\ is 1.68 [27] and using these σ* values and the relationship pK a = 15.9-1.42 Σσ* [28] we estimate that the pK a of the oxyanion of the hemiacetal formed when chymotrypsin and Z-Ala-Ala-Phe-CHO react will be 12.6. Therefore chymotrypsin has lowered the pK a of the larger hemiacetal-1 (Fig. 1c ) by 12.6-7.0 = 5.6 pK a units and the pK a of the smaller hemiacetal-2 (Fig. 1c) by a similar amount ( Table 4 ). The oxyanions of both hemiacetals are therefore both stabilized bỹ 32 kJ mol −1 (Table 4 ).

With the glyoxal inhibitor (ZAAF-COCHO) there is a~40 fold decrease in binding when the pH is decreased from 7.2 to 3.2 and there is a smaller~2.6 fold decrease in binding on increasing the pH from 7.2 to 10.6 ( Table 5 ). However, with the aldehyde inhibitor (ZAAF-CHO) there is a much smaller decrease in binding of~3 when the pH is decreased to from 7.2 to 3.2 (Table 5 ). However, from pH 7.2 to 10.6 there is an~3 fold decrease in binding for both aldehydes and glyoxals (Table 5) . A similar small 4 fold increase in K i from pH 8 to 3 with Bz-Phe-CHO and chymotrypsin has led to the suggestion that the hemiacetal is neutral throughout the pH range 3-8 [11, 15] . As our results show that oxyanion formation occurs according to a pKa of~7 then we conclude that this suggestion is incorrect and the hemiacetal is not neutral at pH 7 or higher.

With ZAAF-COCHO hemiketal formation (K HKobs ) is unchanged from pH 7.2 to 3.2 (Table 5) However, as a result of the better binding of the aldehyde compared to the glyoxal at pH 3.2 there is a~2 fold increase in hemiacetal formation (K HAobs ) from pH 7.2 to 3.2 with ZAAF-CHO (Table 5 ). In contrast with ZAPF-COCHO hemiketal formation decreases from pH 7.2 to 3.2 or 10.2 [4] .

The binding of ZAAF-H decreased 6 fold from pH 7.2 to 10.2 and both aldehyde and glyoxal binding decreased~3 fold from pH 7.2 to 10.2 and for the hydrate signal in (A) the fitted parameters were pKa = 13.23 ± 0.01, δ 1 = 91.04 ± 0.01 ppm and δ 2 = 95.28 ± 0.24 ppm. For the hemiacetal signal in (C) the fitted parameters were pK a = 7.00 ± 0.16, δ 1 = 99.25 ± 0.02 ppm and δ 2 = 100.14 ± 0.02. ppm. For the hemiacetal signal in (B) the continuous line was calculated using the equation δ obs = (δ 1 [H] 2 + δ 2 K a1 [H] + K a1 K a2 K a3 )/([H] 2 + K a1 [H]) + K a1 K a2 ) and the fitted parameters pK a1 = 6.86 ± 0.05, pK a2 = 10.65 ± 0.02, δ 1 = 95.55 ± 0.02 ppm, δ 2 = 97.66 ± 0.02 ppm and δ 3 = 98.99 ± 0.04 ppm. Table 3 Titration constants from pH dependent changes in the chemical shifts of the 13 C-enriched carbons of Z-Ala-Ala-Phe- 13 CHO and Z-Ala-Ala-Phe- 13 ( Table 5 ). Similar decreases were observed with ZAPF-H and ZAPF-COCHO [4] supporting the suggestion [16] that this increase is due to a conformational change resulting from the ionization of the isoleucine-16/aspartate-194 ion pair in chymotrypsin.

In chymotrypsin signals at~18 and~13 ppm have been assigned to the Nδ1 and Nε2 protons of the positively charged imidazolium ring of histidine-57 and the signal at~15 ppm has been assigned to the Nδ1 proton of the neutral imidazole ring [16, [29] [30] [31] [32] . In the glyoxal complex of α-chymotrypsin with Z-Ala-Ala-Phe-COCHO the signals at~18 ppm and~13 ppm are present at both low and high pHs at 25°C demonstrating that the binding of the glyoxal inhibitor has raised the pKa of the active site histidine to a value N 11 [16] . In contrast, in the aldehyde complex of α-chymotrypsin and Z-Ala-Ala-Phe-CHO the signals were barely discernable at 25°C (Fig. 3A) and they were similar to those observed from free α-chymotrypsin under the same conditions (Fig. 3B) . However, at 4°C the signals at 17.9 ppm and~13.0 ppm were clearly visible at low pH, both in the presence (Fig. 3C1) and absence (Fig. 3D1 ) of Z-Ala-Ala-Phe-CHO. Unlike with the analogous glyoxal inhibitor [16] these signals disappeared on increasing the pH and were replaced by a single large signal at 14.7 ppm at pHs N 9.4 and 4°C (Fig. 3C8 ). This signal (Fig. 3C8 ) was similar to those observed with free α-chymotrypsin at 4°C (Fig. 3D8) . These signals were barely visible at 25°C (Fig. 3A8,B8 ). This shows that there is more exchange broadening with the aldehyde inhibitor compared to its glyoxal analogue. Therefore the exchange rates are faster with the aldehyde inhibitor than with the glyoxal inhibitor. It also shows that with the aldehyde inhibitor the histidine pKa must be in the range 5-9. These spectra and the glyoxal inhibitor spectra were obtained using the Watergate W5 pulse sequence [20] . The 1331 water suppression pulse sequence [21] is effective over a smaller chemical shift range of 14-20 ppm, but it is more effective at detecting exchange broadened signals [14] . Therefore we have used the 1331 pulse sequence to follow the ionization of the imidazolium ion of histidine-57 by the pH titration of the Nδ1 proton from~18 to~15 ppm in the presence and absence of Z-Ala-Ala-Phe-CHO at 25 and 4°C (Fig. 4) . From the pH dependence of the chemical shifts of the H-bonded protons (Fig. 5 ) the histidine-57 pK a values in free chymotrypsin and in the Z-Ala-Ala-Phe-CHOchymotrypsin complex were 7.32 ± 0.18 (Fig. 5D) and 7 .97 ± 0.20 (Fig. 5C ) respectively at 4°C. Similar values of 7.35 ± 0.09 and 7.87 ± 0.18 were obtained for the free chymotrypsin (Fig. 5B ) and the Z-Ala-Ala-Phe-CHO-chymotrypsin complex (Fig. 5A) respectively at 25°C. The histidine pK a value for the free enzyme is similar to the values of 6.7 and 7.5 reported for αand δ-chymotrypsin respectively [16, 32] . The increase in the histidine pK a from 7.3 to 7.9 on adding Z-Ala-Ala-Phe-CHO to α-chymotrypsin contrasts with the observed decrease in the histidine pKa to 6.5 when Acetyl-Leu-Phe-CHO binds to α-chymotrypsin [14] .

Aldehydes have been widely used as potent inhibitors of the serine proteases [1] [2] [3] 33] and our results show that the aldehyde group can increase inhibitor potency by a factor of 100 (Table 1 ). The potency of this inhibition is usually attributed to the ability of the aldehyde group to react with active site serine hydroxyl group to form a hemiacetal that mimics the tetrahedral intermediate formed during substrate catalysis [6, 15] . The fact that the aldehyde group of ZAAF-CHO is readily hydrated (K H1obs = 13.9) illustrates how readily hydroxyl groups react with aldehyde carbonyl carbon to give a tetrahedral species. However, solvent water hydroxyl groups compete with the enzymes catalytic serine hydroxyl decreasing the concentration of the non-hydrated aldehyde group which reacts with hydroxyl group of the catalytic serine residue. Therefore the observed K i values are normally corrected to allow for this using K iobs = K i (1 + K HYD ). In our case the K i value for ZAAF-CHO would be decreased by a factor of 14.9 to 0.11 μM. For ZAAFCOCHO we have shown that the active site serine hydroxyl group of chymotrypsin reacts with the keto carbonyl of the glyoxal group which has a lower K H1obs = 1.58 therefore the observed K i will only be reduced by a factor of 2.58 to 0.14 μM which is similar to the corrected K i value of 0.11 μM obtained with the aldehyde inhibitor. Therefore the fact that glyoxal inhibitors are bound~4 fold more tightly than the corresponding aldehyde inhibitor is due to the fact that with the glyoxal inhibitors hemiketal formation occurs at the less effectively hydrated keto carbon as opposed to the more heavily hydrated aldehyde carbon.

Inhibition of chymotrypsin by glyoxal inhibitors has been shown to be stereospecific with only one diastereomeric hemiketal being formed [16, 17] . However, with the corresponding peptide aldehyde inhibitor, inhibition is not fully stereospecific with two diastereoisomers being formed. The diastereoisomer at 95.5 ppm predominates (~75%) with the diastereoisomer at 99.2 ppm making up the remaining~25% of the hemiacetal formed. Two diastereoisomers were also observed when N-Acetyl-Phe-CHO was incubated with chymotrypsin but the proportions and chemical shifts were not reported [12] . Likewise when Leupeptin was incubated with trypsin two similar diastereoisomers were detected with chemical shifts of 98.8 ppm (86%) and 97.2 ppm (14%) [13] . These titrated with pK a values of 4.69 and 5.67. In the present work both the chymotrypsin diastereoisomers formed with Z-Ala-Ala-Phe-CHO titrated but their Table 4 Hemiacetal and hemiketal oxyanion stabilization in aldehyde (ZAAF-CHO) and glyoxal (ZAAF-COCHO) inhibitor complexes formed with α-chymotrypsin.

Inhibitor complexed with chymotrypsin Oxyanion pK a pK a decrease Oxyanion stabilization pK aw (water) pK ae (enzyme) pK aw -pK ae ΔG (kJ mol −1 )

Hemiacetal-1 ZAAF-13 CHO 12.6 a 7.0 b 5.6 32.0 Hemiacetal-2 ZAAF-13 CHO 12.6 a 6.9 c 5.7 32.5 Hemiketal ZAAF 13 COCHO 9.5 d 4.6 e 4.9 28.0 a pK a of hemiacetal in water calculated as described in the text. b pK a of hemiacetal-1 formed with α-chymotrypsin as in Scheme 1. c pK a of hemiacetal-2 formed with α-chymotrypsin as in Scheme 1. d pK a of the hemiketal in water calculated as described for the hemiacetal except that a substituent constant, σ* = 2.15 was used for the aldehyde group of the glyoxal inhibitor [25] . e pK a of hemiketal formed with chymotrypsin [16] . pK a values were very similar at 6.9 and 7.0 (Table 4 ) suggesting that they are both in similar environments. X-ray crystallographic studies of aldehyde inhibitors binding to serine proteases [14, 34, 35] has shown that hemiacetal formation is not stereospecific with aldehyde inhibitors with two diastereomeric hemiacetals being formed. One of these has the oxyanion in the oxyanion hole while in the other diastereoisomer the oxyanion points towards the active site histidine [14, 34, 35] and is hydrogen bonded to Nε2 of histidine-57. The oxyanion in the oxyanion hole is about 2.8 Å from the backbone amide nitrogens of serine-195 and glycine-193. However, in the other diastereoisomer the oxyanion is about 2.9 Å from the Nε2 nitrogen of the positively charged imidazolium ion of histidine-57. Therefore the oxyanions are in different electrostatic environments in the two diastereoisomers and so we would expect that they would have significantly different pK a values as is observed for the two diastereoisomeric hemiacetals observed in the Acetyl-Leu-Phe-CHO/trypsin complex [14] . The fact that in the Z-Ala-Ala-Phe-CHO/chymotrypsin complex the oxyanion pK a values were~7 for both diastereoisomers suggests that the oxyanions in both diastereoisomers are in similar electrostatic environments.

Since the binding Z-Ala-Ala-Phe-CHO raised the pK a of histidine-57 to~8 (Fig. 5) and the oxyanion pK a values of the diastereomeric hemiacetals were both~7 (Fig. 2) it is expected that the positively charged histidine residue will play an important role in lowering the oxyanion pK a to~7. The binding of Acetyl-Leu-Phe-CHO to α-chymotrypsin resulted in the formation of a hemiacetal signal at 102 ppm which did not titrate from pH 7 to 13 [14] . The chemical shift value of 102 ppm is larger than that observed (~100 ppm) when the oxyanion with Z-Ala-Ala-Phe-CHO is fully formed (Fig. 2B,C) . This suggests that the oxyanion is fully formed at pH 7 with Acetyl-Leu-Phe-CHO and so should have a pK a b 6. The histidine pK a of 6.5 in the Acetyl-Leu-Phe-CHO complex [14] shows that it should be able to stabilize the oxyanion. However, with glyoxal inhibitors the histidine pKa is raised to N 11 therefore it is clear that while aldehyde inhibitors like glyoxal inhibitors are bound tightly to chymotrypsin, they are much less effective at raising the pKa of the active site histidine in chymotrypsin. Although oxyanion stabilization is more effective with the aldehyde hemiacetal oxyanion than with the glyoxal hemiketal oxyanion ( Table 4 ) the pKa of the imidazolium ion of histidine-57 is only raised to~8 with hemiacetal formation (Table 6 ) while it is raised to N 11 with hemiketal formation in glyoxal complexes [16] inhibitor and to N 10 when peptidyl trifluoromethyl ketones [36] or peptidyl boronic acid [37] inhibitors form negatively charged tetrahedral adducts with chymotrypsin. This shows that oxyanion formation only has a minor role in raising the pKa of the imidazolium ion of histidine-57 in aldehyde inhibitor complexes. Therefore replacing the aldehyde hydrogen with an aldehyde group [16, 17] or trifluoromethylene group [38] is the major factor in strengthening the hydrogen bond between histidine-57 and aspartate-102, raising the pK a of the imidazolium ion of histidine-57 and ensuring that tetrahedral intermediate formation is stereospecific. Similar effects are expected during catalysis with substrates.

Therefore tetrahedral adduct formation only increases the strength of the hydrogen bond between the aspartate and histidine residues raising the pK a of the imidazolium ion of histidine-57 to a value N 10 if the leaving group peptide nitrogen is present or if an inhibitor carbon atom is present in an equivalent position. This suggests that the inhibitor aldehyde hydrogen atom is not large enough to compress histidine-57-aspartate-32 hydrogen bond and/or screen it from solvent so that the histidine pK a is raised to a value N 10. Likewise the aldehyde proton must be replaced by a larger group to ensure that the formation of the tetrahedral species is stereospecific.

The catalytic serine group is expected to have a pK a of~15 and so for the imidazolium ion of histidine-57 to act as an effective base catalyst its pKa should be raised from its value of 7 in the free enzyme to a value similar to that of the active site serine hydroxyl group when a substrate 17.85 ± 0.06 ppm and δ 2 = 14.84 ± 0.06 ppm, (B) pK a = 7.35 ± 0.09, δ 1 = 18.11 ± 0.05 ppm and δ 2 = 14.73 ± 0.04. ppm, (C) pK a = 7.97 ± 0.20, δ 1 = 17.79 ± 0.08 ppm and δ 2 = 14.54 ± 0.08 ppm and (D) pK a = 7.32 ± 0.18, δ 1 = 18.01 ± 0.12 ppm and δ 2 = 14.56 ± 0.09 ppm. Table 6 Titration constants from pH dependent changes in the chemical shift of the N δ1 proton of histidine-57 in α-chymotrypsin and in the Z-Ala-Ala-Phe-CHO/α-chymotrypsin complex at 4 and 25°C. binds [4, 22] . If the aldehyde inhibitor has only a small effect on the pK a of the active site histidine before tetrahedral adduct formation, then the fact that the effective molarity of the hydroxyl group of the catalytic serine group is only~7× lower with the aldehyde inhibitor compared with its equivalent glyoxal inhibitor (Table 2) shows that a large increase in the pK a of histidine-57 is not required to increase the reactivity of the serine hydroxyl group for formation of the tetrahedral hemiacetal. This suggests that the expected increase in the effective molarity of the active site serine hydroxyl required for tetrahedral intermediate formation during catalysis cannot be solely attributed to a large increase in the pK a of histidine-57. Therefore we propose that the primary reason for the increase in the effective molarity of the catalytic hydroxyl when it binds substrates will be the entropic advantage achieved by optimally aligning the catalytic serine's hydroxy group with the carbonyl carbon of the peptide being hydrolysed. We conclude that a moiety larger than the aldehydic proton must be present if the pK a of the active site histidine is to be raised to a value N10 and if there is to be stereospecific formation of a tetrahedral adduct. With aldehyde inhibitors there is stabilization of the hemiacetal oxyanion with its pK a being reduced by~5.6 pK a units while the pK a of the active site histidine is only raised~0.5 pKa units. Therefore we conclude that a large increase in the pK a of the active site histidine is not required for oxyanion stabilization or for the active site serine hydroxyl group to have an effective molarity of 6000. As the active site serine hydroxyl group is expected to have a pKa~15 then we have argued [4, 16] that the large increase in the pKa of the active site histidine (i.e. pKa N 11) observed on binding glyoxal inhibitor is required for it to increase the reactivity of the serine hydroxyl group by general acid catalysis. But, with the aldehyde inhibitor there is only a small increase in the pKa of the active site histidine pKa to~8. However, the effective molarity of the serine hydroxyl group with the aldehyde inhibitor is only~7 fold smaller than that observed with the corresponding glyoxal inhibitor ( Table 2 ). This clearly demonstrates that only a small part of the high reactivity of the serine hydroxyl group can be attributed to general base catalysis by the catalytic histidine residue of chymotrypsin.

Prostate-specific antigen is a "chymotrypsin-like" serine protease with unique P1 substrate specificity

Studies of the synthesis of all stereoisomers of MG-132 proteasome inhibitors in the tumor targeting approach

Structure-based design, synthesis, and evaluation of peptidemimetic SARS 3CL protease inhibitors

Importance of tetrahedral intermediate formation in the catalytic mechanism of the serine proteases chymotrypsin and subtilisin

Aldehydes as inhibitors of papain

Use of peptide aldehydes to generate transition-state analogs of elastase

Evidence for hemiacetal formation between N-acyl-L-phenylalaninals and alpha-chymotrypsin by cross-saturation nuclear magnetic resonance spectroscopy

Evidence for hemiacetal formation between alpha-chymotrypsin and hydrocinnamaldehyde by cross-saturation nuclear magnetic resonance spectroscopy

A proton-magnetic-resonance study of N-trifluoroacetyl-L-alanyl-L-phenylalaninal binding to alpha-chymotrypsin

Proton and fluorine nuclear magnetic resonance spectroscopic observation of hemiacetal formation between N-acyl-p-fluorophenylalaninals and alpha-chymotrypsin

Fluorine nuclear magnetic resonance spectroscopy of "transition state analogue" complexes of the D and L enantiomers of N-Acetyl-pfluorophenylalinal and alpha-chymotrypsin

13C NMR spectroscopy of "transition-state analogue" complexes of N-acetyl-L-phenylalaninal and alpha-chymotrypsin

Diastereotopic covalent binding of the natural inhibitor leupeptin to trypsin: detection of two interconverting hemiacetals by solution and solid-state NMR spectroscopy

Correlation of low-barrier hydrogen bonding and oxyanion binding in transition state analogue complexes of chymotrypsin

Mechanism of association of a specific aldehyde "transition-state analogue" to the active site of alpha-chymotrypsin

13 C and 1 H NMR studies of ionizations and hydrogen bonding in chymotrypsin-glyoxal inhibitor complexes

A 13 C-NMR study of the inhibition of delta-chymotrypsin by a tripeptide-glyoxal inhibitor

An NMR study of the inhibition of pepsin by glyoxal inhibitors: mechanism of tetrahedral intermediate stabilization by the aspartyl proteinases

A 13 C-N.M.R. investigation of the ionizations within an inhibitor-alpha-chymotrypsin complex: evidence that both alpha-chymotrypsin and trypsin stabilize a hemiketal oxyanion by similar mechanisms

Improved WATERGATE pulse sequences for solvent suppression in NMR spectroscopy

Solvent suppression in Fourier transform nuclear magnetic resonance

A study of the stabilization of tetrahedral adducts by trypsin and delta-chymotrypsin

Structure and stereochemistry of tetrahedral inhibitor complexes of papain by direct NMR observation

Equilibrium and rate constants for the interconversion of two conformations of α-chymotrypsin. The existence of a catalytically inactive conformation at neutral pH

pKa Prediction for Organic Acids and Bases

Calculation of the pKa values of alcohols from σ* Constants and from the carbonyl frequencies of their esters

Oxyanion and tetrahedral intermediate stabilization by subtilisin: detection of a new tetrahedral adduct

Acid ionization constants of alcohols. II. Acidities of some substituted methanols and related compounds

Confirmation of the assignment of the low-field proton resonance of serine proteases by using specifically nitrogen-15 labeled enzyme

Protonation-state dependence of hydrogen bond strengths and exchange rates in a serine protease catalytic triad: bovine chymotrypsinogen A

High resolution nuclear magnetic resonance study of the histidine-aspartate hydrogen bond in chymotrypsin and chymotrypsinogen

High resolution nuclear magnetic resonance studies of the active site of chymotrypsin

Slow-binding inhibition of chymotrypsin and cathepsin G by the peptide aldehyde chymostatin

The 1.8 Å structure of the complex between chymostatin and Streptomyces griseus protease A. A model for serine protease catalytic tetrahedral intermediates

Two crystal structures of the leupeptin-trypsin complex

Inhibition of papain by nitriles: mechanistic studies using NMR and kinetic measurements

Proton magnetic resonance studies of the active center histidine of chymotrypsin complexed to peptideboronic acids: solvent accessibility to the N-delta and N-epsilon sites can differentiate slow-binding and rapidly reversible inhibitors

Complex of α-chymotrypsin and N-acetyl-L-leucyl-L-phenylalanyl trifluoromethyl ketone: structural studies with NMR spectroscopy

This work was supported by the Irish Research Council and University College Dublin. Grant nos. 055637/Z/98 from the Wellcome Trust was used to purchase the NMR spectrometer used in these studies and Science Foundation Ireland funding was used to upgrade the NMR spectrometer.