Hemiacetal stabilization in a chymotrypsin inhibitor complex and the reactivity of the hydroxyl group of the catalytic serine residue of chymotrypsin

Article abstract of DOI:10.1016/j.bbapap.2014.03.008

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 pK_a value of ～ 7 showing that chymotrypsin reduces the oxyanion pK_a values by ～ 5.6 pK _a units and stabilizes the oxyanions of both diastereoisomers by ～ 32 kJ mol^{- 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 pH 7.2 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 pK_a 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 pK_a and that the aldehyde hydrogen must be replaced by a larger group to raise the histidine pK_a > 10 and give stereospecific formation of tetrahedral intermediates. The results show that a large increase in the pK_a of the active site histidine is not needed for the active site serine hydroxyl group to have an effective molarity of 6000.

Full text of DOI:10.1016/j.bbapap.2014.03.008

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Biochimica et Biophysica Acta xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
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Hemiacetal stabilization in a chymotrypsin inhibitor complex and
thereactivity of the hydroxyl group of the catalytic serine residue
of chymotrypsin
4Q1 Jennifer A. Cleary a, William Doherty b, Paul Evans b, J.Paul G. Malthouse a,
⁎
a
5
6
School of Biomolecular and Biomedical Science, Centre for Synthesis and Chemical Biology, Conway Institute, University College Dublin, Dublin 4, Ireland
School of Chemistry and Chemical Biology, Centre for Synthesis and Chemical Biology, Conway Institute, University College Dublin, Dublin 4, Ireland
b
OOF
7
a r t i c l e i n f o
a b s t r a c t
The aldehyde inhibitor Z-Ala-Ala-Phe-CHO has been synthesized and shown by 13C-NMR to react with the active 19
site serine hydroxyl group of alpha-chymotrypsin to form two diastereomeric hemiacetals. For both hemiacetals 20
oxyanion formation occurs with a pKa value of ~7 showing that chymotrypsin reduces the oxyanion pKa values by 21
~5.6 pKa units and stabilizes the oxyanions of both diastereoisomers by ~32 kJ mol−1. As pH has only a small 22
effect on binding we conclude that oxyanion formation does not have a significant effect on binding the aldehyde 23
inhibitor. By comparing the binding of Z-Ala-Ala-Phe-CHO with that of Z-Ala-Ala-Phe-H we estimate that the 24
aldehyde group increases binding ~100 fold. At pH 7.2 the effective molarity of the active site serine hydroxy 25
group is ~6000 which is ~7× less effective than with the corresponding glyoxal inhibitor. Using 1H-NMR we 26
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 27
Z-Ala-Ala-Phe-CHO is bound. We conclude that oxyanion formation only has a minor role in raising the histidine 28
pKa and that the aldehyde hydrogen must be replaced by a larger group to raise the histidine pKa N 10 and give 29
stereospecific formation of tetrahedral intermediates. The results show that a large increase in the pKa of the ac- 30
tive site histidine is not needed for the active site serine hydroxyl group to have an effective molarity of 6000. 31
© 2014 Published by Elsevier B.V. 32
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Article history:
Received 12 December 2013
Received in revised form 13 February 2014
Accepted 14 March 2014
Available online xxxx
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Keywords:
Chymotrypsin
Aldehyde inhibitor
Hemiacetal
Oxyanion
Effective molarity
36
34
35
37
1. Introduction
The rate-limiting step in peptide catalysis by the serine proteases is 53
thought to be either formation or breakdown of a tetrahedral interme- 54
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51
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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 po-
tent 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
UNCORRECTED PR
determine how the aldehyde group contributes to inhibitor potency
with chymotrypsin.
diate formed by the addition of the hydroxyl group of the catalytic ser- 55
ine to the carbonyl carbon of the peptide bond being hydrolysed. The 56
tight binding of aldehydes to the cysteine protease papain was attribut- 57
ed to the formation of a thiohemiacetal by the addition of the thiolate 58
ion of the catalytic cysteine to the carbonyl of the peptide bond being 59
hydrolysed analogous to the tetrahedral intermediate formed during 60
catalysis [5]. Similar proposals were made when aldehydes were 61
found to be potent inhibitors of the serine protease elastase [6]. Using 62
1H-NMR indirect evidence for hemiacetal formation with the serine 63
protease chymotrypsin was obtained using saturation transfer [7–9]. Di- 64
rect observation of a hemiacetal signal was reported using 19F-NMR 65
with N-Acetyl-p-fluorophenylalinals and α-chymotrypsin [10,11]. 13C- 66
NMR studies of the aldehyde inhibitor Acetyl-Phe-CHO binding to α- 67
chymotrypsin have shown two hemiacetals are formed [12]. 13C-NMR 68
has shown that the aldehyde inhibitor leupeptin (N-Acetyl-Leu-Leu- 69
Leu-Arg-CHO) also forms two diastereomeric hemiacetals with trypsin 70
[13]. However, with Acetyl-Leu-Phe-CHO it was reported that only 71
one hemiacetal signal was observed [14]. The fact that only small chang- 72
es (≤4 fold) in Ki values have been observed from pH 3–8 when alde- 73
hyde inhibitors bind to chymotrypsin, has led to the suggestion that 74
the hemiacetal formed with chymotrypsin remains neutral from 75
pH 3–8 [11,15]. Likewise, the fact that the chemical shifts of the 76
Abbreviations: Z, benzyloxycarbonyl
⁎
Corresponding author at: UCD School of Biomolecular and Biomedical Science, UCD
Centre for Synthesis and Chemical Biology, Conway Institute, University College Dublin,
Dublin 4, Ireland. Tel.: +353 17166872; fax: +353 17166893.
E-mail address: J.Paul.G.Malthouse@ucd.ie (J.P.G. Malthouse).
http://dx.doi.org/10.1016/j.bbapap.2014.03.008
1570-9639/© 2014 Published by Elsevier B.V.
Please cite this article as: J.A. Cleary, et al., Hemiacetal stabilization in a chymotrypsin inhibitor complex and thereactivity of the hydroxyl group of
the catalytic..., Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.03.008

2
J.A. Cleary et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
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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 13C-NMR to deter-
mine 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].
and to determine the effective molarity of the hydroxyl group of the 93
catalytic serine residue in these complexes [4]. In this study we use a 94
similar approach to quantify hemiacetal formation in a chymotrypsin– 95
aldehyde inhibitor complex and to determine the effective molarity of 96
the hydroxyl group of the catalytic serine residue in this complex. We 97
also undertake an extensive 1H-NMR study of how the ionization state 98
of the active site histidine residue changes when aldehyde inhibitors 99
are bound. This allows us to determine how oxyanion formation affects 100
the histidine pKa and also whether a high histidine pKa is required for 101
the active site serine hydroxyl group to have a high effective molarity. 102
Finally we discuss the mechanistic significance of these results.
103
104
105
2. Materials and methods
Recently we have developed a procedure that allows us to quantify
hemiketal formation in chymotrypsin–glyoxal inhibitor complexes
2.1. Materials
L-[1-13C]Phenylalanine (99 at.%) was obtained from Cambridge 106
Isotope laboratories, Inc. (50 Frontage Road, Andover, MA 01810-5413 107
USA). All other chemicals were obtained from Sigma-Aldrich Chemical 108
co., Gillingham, Dorset, U.K.
109
2.2. Synthesis of Z-Ala-Ala-Phe-H, Z-Ala-Ala-Phe-COOCH3 and
Z-Ala-Ala-Phe-COCHO
110
111
Z-Ala-Ala-Phe-H (Scheme 1A) was synthesized as described by 112
Petrillo et al. [4]. Z-Ala-Ala-Phe-13COOCH3 and Z-Ala-Ala-Phe-13COCHO 113
(Scheme 1D) were synthesized as described by Cosgrove et al.,[18].
114
2.3. Synthesis of Z-Ala-Ala-Phe-13CH2OH
115
Z-Ala-Ala-Phe-13COOCH3 (220 mg, 0.48 mmol, 1 equiv.) was sus- 116
pended in anhydrous tetrahydrofuran (2 mL) and added to a stirred sus- 117
pension of LiAlH4 (125 mg, 3.29 mmol, 7 equiv.) in anhydrous THF (3.5 118
mL) under nitrogen at 0 °C. The reaction mixture was left to stir for 1 h 119
at 0 °C followed by a further 30 min at room temperature. The reaction 120
mixture was cooled to 0 °C and ethyl acetate (5 mL) was cautiously 121
added dropwise to the mixture, followed by dropwise addition of ice 122
cold water (5 mL). The resulting mixture was stirred for a further 123
30 min and the layers partitioned. The aqueous layer was extracted 124
with ethyl acetate (2 × 10 mL) and the combined organic layers were 125
washed with brine (10 mL), dried over MgSO4, filtered and solvent 126
was removed in vacuo to give the crude product. Purification by silica 127
chromatography using CH2Cl2-MeOH; 15:1 as an eluant gave the prod- 128
uct as a white solid. M.p. 170–175 °C.
129
The yield was 0.112 g (0.248 mmol 54%). 13C-NMR analysis gave 130
the following data. δC (100 MHz, d6-DMSO) 18.1 (C, CHCH3), 18.5 131
(C, CHCH3), 36.3 (1C, C6H5CH2), 48.2 (1C, CHCH3), 50.0 (1C, CHCH3), 132
52.4 (1C, C6H5CH2CH), 62.1 (13CH2OH), 65.4 (1C, O\CH2Ph), 125.9– 133
129.1 (10C, CH_CH), 137.0 (1C, CH_C_), 137.5(1C, CH_C_), 155.7 134
(1C, O\CO\NH), 171.6 (1C, CONH), 172.0 (1C, CONH).
135
2.4. Synthesis of Z-Ala-Ala-Phe-13CHO
136
A
sonicated suspension of Z-Ala-Ala-Phe-13CH2OH (94 mg, 137
0.22 mmol, 1 equiv.) in anhydrous CH2Cl2 (3 mL) was added to a stirred 138
solution of pyridinium chlorochromate (315 mg, 1.46 mmol, 6.5 equiv.) 139
and Celite (280 mg) in anhydrous CH2Cl2 (2 mL). The reaction mixture 140
was left to stir for 6 h after which time silica (ca. 2 g) was added and 141
solvent was removed in vacuo. The residue was purified by silica chro- 142
matography using ethylacetate as an eluant. This gave the product as a 143
white solid.
144
The yield was 0.038 g (0.085 mmol, 41%). 13C-NMR analysis gave 145
the following data. δC (100 MHz, d6-DMSO) 18.1 (C, CHCH3), 18.4 (C, 146
CHCH3), 33.2 (1C, C6H5CH2) 47.9 (1C, CHCH3), 49.9 (1C, CHCH3), 147
59.5 (1C, C6H5CH2CH), 65.3 (1C, O\CH2Ph), 89.8 (1C, \13CH(OH)2), 148
126.3–129.2 (10C, CH_CH), 137.0 (1C, CH_C_), 137.5(1C, CH_C_), 149
Scheme 1. Structure of inhibitors. R is ZAA (Z-Ala-Ala).
Please cite this article as: J.A. Cleary, et al., Hemiacetal stabilization in a chymotrypsin inhibitor complex and thereactivity of the hydroxyl group of
the catalytic..., Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.03.008

J.A. Cleary et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
3
150 155.7 (1C, O\CO\NH), 171.0 (1C, CONH), 172.7 (1C, CONH), 200.2
151 (1C, \13CHO)
152 2.5. Chymotrypsin solutions
153
α-Chymotrypsin (crystallized and lyophilized) was obtained from
154 the Sigma Chemical Co. The amount of fully active enzyme (~77%)
155 was determined as described by Finucane et al. [19].
156 2.6. Determination of Ki values
157
The Ki value for inhibition of the chymotrypsin-catalyzed hydrolysis
158 of succinyl-Ala-Ala-Pro-Phe-pna at pH 7.2 in 0.1 M potassium phos-
159 phate buffer was determined by determining KM(obs) values at different
160 inhibitor concentrations and then plotting KM(obs) versus the inhibitor
161 concentration. Ki was determined from the intercept on the x-axis. All
162 other Ki values were determined when [S0] ≪ KM by as described by
163 [4].
164 2.7. NMR spectroscopy
Fig. 1. Effect of pH on the 13C-NMR spectra from the Z-Ala-Ala-Phe-13CHO in the presence
of α-chymotrypsin. 13C-NMR acquisition and processing parameters were as described in
OOF
165
NMR spectra at 11.75 T were recorded with a Bruker Avance DRX
the Materials and methods section. Sample conditions were (a) 3.0 ml, 0.2 mM Z-Ala-Ala-
Phe-13CHO, 1.7% (v/v) d6-DMSO, 1 mM HCl, (b) 3.0 ml, 1.7 mM α-chymotrypsin. Samples
(c)–(h) were 3.0 ml and contained 1.7 mM α-chymotrypsin, 2.1 mM Z-Ala-Ala-Phe-
13CHO, 1.8% (v/v) d6-DMSO. Samples (i) and (j) were 3.1 ml and contained 1.7 mM
α-chymotrypsin, 2.0 mM Z-Ala-Ala-Phe-13CHO, 1.8% (v/v) d6-DMSO. All samples
contained 10% 2H2O which was used as a deuterium lock signal and samples (b)–(h)
also contained ~10 mM potassium phosphate to help maintain stable pH values.
166 500 standard-bore spectrometer operating at 125.7716 MHz for 13C-
167 nuclei. 10 mm-diameter NMR tubes containing ~3 mL samples were
168 used for 13C-NMR spectroscopy. The 13C-NMR spectral conditions
169 for the samples were: 8192 time domain data points; spectral width
170 236.6 ppm; acquisition time 0.138 s; 0.6 s relaxation delay time; 90 °C
171 pulse angle; 2048 transients recorded per spectrum. Waltz-16 compos-
172 ite pulse 1H decoupling of 1.0 W was used which was reduced to
173 0.026 W during the relaxation delay to minimize dielectric heating
174 but maintain the Nuclear Overhauser Effect.
values of 0.64 0.07 s and 0.69 0.08 s respectively and are assigned 205
to diastereomeric hemiacetals formed when the active site serine hy- 206
droxyl group reacts with the 13C-enriched inhibitor aldehyde group. A 207
hydrate cannot form diastereoisomers and would only give one NMR 208
signal. Therefore the fact that we observe two 13C-NMR signals confirms 209
that two diastereomeric hemiacetals have been formed and that the in- 210
hibitor is not bound as a hydrate. The inhibitor was tightly bound at 211
both acid and alkaline pHs (Table 5) and we have calculated that 212
more than 98% of the enzyme is saturated with inhibitor from pH 3.2 213
175
1H-NMR spectra were recorded at 500 MHz using 5-mm-diameter
176 NMR tubes. Spectral conditions used for observing H-bonded protons
177 at 12–18 ppm were: 32768 time domain data points, spectral width
178 40 ppm, acquisition time 0.819 s, 1.0 s relaxation delay, 90° pulse
179 angle, 4 dummy scans and 512 transients were recorded per spectrum.
180 Water suppression was carried out with the Watergate W5 pulse se-
181 quence [20] and the 1331 pulse sequence [21].
182
All spectra were transformed using an exponential weight factor of
to 10.6.
214
183 50 Hz. Both 1H and 13C chemical shifts are quoted relative to tetra-
184 methylsilane at 0.00 ppm. The chemical shift of d6-dimethyl sulfoxide
185 at 38.7 ppm was used as a secondary reference for 13C-NMR spectra ob-
186 tained in aqueous solutions. Aqueous samples contained 10% (v/v) 2H2O
187 to obtain a deuterium lock signal.
Hemiacetals like hemiketals are expected to have chemical shifts 215
~4 ppm larger than their hydrates [16]. Therefore the signal at 216
95.5 ppm has a chemical shift close to that expected if the hydroxyl 217
group of the active site serine of chymotrypsin forms a hemiacetal 218
with the inhibitor aldehyde group. If this process is stereospecific 219
then only one hemiacetal signal will be observed. However, as two 220
hemiacetal signals are observed then we can conclude that hemiac- 221
188 3. Results
etal formation is not stereospecific.
From the areas of signals at 99.2 ppm and 95.5 ppm (Fig. 1c) the 223
proportions of the corresponding hemiacetals at pH 2.93 were 22.3 224
222
189 3.1. 13C-NMR spectra of Z-Ala-Ala-Phe-13CHO in an aqueous solution
190
In an aqueous solvent at pH 7.2 containing 10 mM sodium phos-
0.6% and 77.7 0.6% respectively. Similar proportions (22.6 2.1% and 225
77.4 2.1%) were obtained at pH 5.85 (Fig. 1e). Due to line broadening 226
accurate measurements could not be made at intermediate pHs. Howev- 227
191 phate buffer, 10% d6-DMSO and 10% 2H2O Z-Ala-Ala-Phe-13CHO
192 (Scheme 1C) gave signals at 202.7 and 91.0 ppm with JCH values of
193 183.0 0.3 Hz and 163.4 0.1 Hz respectively. T1 (spin lattice relaxa-
194 tion times) were determined by the inversion recovery method. The sig-
er, 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
228
229
UNCORRECTED PR
195 nal at 91 ppm had a spin lattice relaxation time of 0.61 0.10 s. Using
3.0%). The mean values are 23
2.4% for the diastereoisomer with a 230
196 quantitative 13C-NMR it was determined that the hydration constant
197 (KH1obs) was 13.9 0.7 (Mean of 13 determinations.).
Table 1
t1:1
t1:2
t1:3
198 3.2. 13C-NMR of α-chymotrypsin inhibited by Z-Ala-Ala-Phe-13CHO
Inhibition of chymotrypsin by peptide aldehyde and peptide glyoxal inhibitors at pH 7.2:
Contribution of the aldehyde and glyoxal groups to inhibition.
On adding Z-Ala-Ala-Phe-13CHO (Fig. 1a) to α-chymotrypsin
Inhibitor
Ki(obs) (μM)
KiZAAF-H/KiZAAF-CHO
KiZAAF-H/KiZAAF-COCHO
t1:4
199
10a
1.7
0.37b
t1:5
t1:6
t1:7
200 (Fig. 1b) new signals were observed at 99.2 ppm (JCH 158.8 Hz) and
201 95.5 ppm (JCH 169.3 Hz) at pH 2.93 (Fig. 1c). The signal at 38.7 ppm is
202 due the d6 = DMSO (1.8% (v/v)) used to make the inhibitor stock solu-
203 tion and the signal at 91.2 ppm (JCH 163.4 Hz) (Fig. 1c) is due to a small
204 excess of the inhibitor (Fig. 1a). The signals at 99.2 and 95.5 had T1
ZAAF-H
ZAAF-CHO
ZAAF-COCHO
170
0.4a
100
460
a
Present work, errors are the standard deviations of 3 or more determinations.
[16]
t1:8
t1:9
b
Please cite this article as: J.A. Cleary, et al., Hemiacetal stabilization in a chymotrypsin inhibitor complex and thereactivity of the hydroxyl group of
the catalytic..., Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.03.008

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J.A. Cleary et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
complex (EAald). KHA1 and KHA2 are the equilibrium constants for the for- 258
mation of hemiacetal-1 and -2 respectively (Scheme 2). The experimen- 259
tally determined disassociation constant Ki(obs) = [E]([Aald] + [Ahyd]) / 260
([EAald] + [Hemiacetal-1] + [Hemiacetal-2]) and so
261
KiðobsÞ ¼ Ksð 1 þ KH1Þ=ð1 þ KHA1 þ KHA2
Þ
ð1Þ
ð2Þ
ð3Þ
263
264
KiðobsÞ ¼ Ksð 1 þ KH1Þ=ð1 þ KHAobs
where KHAobs ¼ KHA1 þ KHA2
Þ
266
267
269
270
n
o
KHAðobsÞ
¼
Ksð1 þ KH1Þ‐KiðobsÞ =KiðobsÞ
ð4Þ
272
Assuming Ki(obs) for ZAAF-H (Scheme 1A) was a good approximation
for the disassociation constant (Ks) of the corresponding chymotrypsin– 273
ZAAF-CHO complex (EAald in Scheme 2) KHA(obs) (Eq. (4)) was calculat- 274
ed. The KHA(obs) of 1490 for hemiacetal formation with ZAAF-CHO was 275
similar in magnitude to KHK(obs) for hemiketal formation with ZAAF- 276
COCHO (Table 2). Using Eq. (3), the value of 1490 for KHK(obs) and the 277
mean proportions of 77% and 23% for the diastereoisomers at 95.5 278
ppm and 99.2 ppm respectively KHA1 and KHA2 were determined for 279
Scheme 2. Minimal scheme for hemiacetal formation by chymotrypsin.
231 signal at 99.2 ppm and 77 2.4% for the diastereoisomer with a signal
232 at 95.5 ppm.
233
Using 13C-NMR two diastereomeric thiohemiacetals were first ob-
the two diastereoisomers (Table 2).
280
234 served when the cysteine protease papain was incubated with an alde-
235 hyde inhibitor [23]. Subsequently two hemiacetals were observed by
236
3.4. Effective molarity of the hydroxyl group of the catalytic serine in 281
α-chymotrypsin 282
13C-NMR when aldehyde inhibitors reacted with the serine proteases
237 chymotrypsin [12] and trypsin [13]. The hemiacetals formed when
238 leupeptin (N-acetyl-Leu-Leu-Arg-CHO) reacted with trypsin [13] had
239 similar chemical shifts and proportions except the signal at higher
240 chemical shift predominated (86%) whereas in the present work the
241 signal at lower chemical shift predominated (77.4–79.2%).
Hemiacetal formation in the chymotrypsin–aldehyde inhibitor com- 283
plex is a first order process (KHAobs = [hemiacetal] / [EA]) whereas the 284
hydration of the free aldehyde inhibitor in water is a second order pro- 285
cess (KH1 = [hemiacetal] / [aldehyde][H2O]). KH1 is calculated by divid- 286
ing KH1(obs) (KH1(obs) = [hemiacetal] / [aldehyde]) by the molarity of 287
water. The ratio KHAobs/KH1 is therefore the effective molarity of the ser- 288
ine hydroxyl group in hemiacetal formation. This effective molarity is 289
the molarity of water required to be as effective as the catalytic serine 290
hydroxyl group in forming a hemiacetal with the inhibitor aldehyde 291
group. The effective molarity of the active site serine hydroxyl group is 292
~7 times lower for hemiacetal formation with ZAAF-CHO (Scheme 1C) 293
compared to hemiketal formation with the keto carbon of the glyoxal 294
242 3.3. Quantifying the role of the aldehyde group in inhibitor binding
243
The Ki value for the inhibition of the chymotrypsin catalyzed hydro-
244 lysis of succinyl-Ala-Ala-Pro-Phe-pna by the aldehyde inhibitor Z-Ala-
245 Ala-Phe-CHO at pH 7.2 was determined from the dependence of the ob-
246 served KM value for succinyl-Ala-Ala-Pro-Phe-pna on the concentration
247 of the inhibitor present. From the Ki values of ZAAF-H and ZAAF-CHO we
248 estimate that the aldehyde group increases binding ~100 fold (Table 1).
group of ZAAF-COCHO (Scheme 1D).
295
249
The glyoxal group of ZAAF-COCHO is bound 4.55 times more tightly
250 than the aldehyde group of ZAAF-CHO (Table 1). 13C-NMR has shown
251 that ZAAF-CHO reacts with chymotrypsin to form two hemiacetals,
252 one smaller signal with a chemical shift of 99.2 ppm and the other larger
253 signal with a chemical shift of 95.5 ppm at pH 2.93 (Fig. 1c). Scheme 2 is
254 the minimal scheme we have used to describe hemiacetal formation.
3.5. Effect of pH on the 13C-NMR signals from Z-Ala-Ala-Phe-CHO in the 296
presence and absence of α-chymotrypsin 297
On increasing the pH the chemical shift of both the large signal at 298
95.5 and the small signal at 99.2 ppm increased (Fig. 1a–j). The chemical 299
shift of the large signal (Fig. 1c) increased from at 95.55 0.02 ppm to 300
97.66 0.02 ppm with a pKa of 6.86 0.05 (Fig. 2B). There was also a 301
further small increase in chemical shift to 98.99 0.04 ppm with a pKa 302
255 KH1obs is the hydration constant for aldehyde inhibitor (KH1(obs)
256
=
hydrate/aldehyde = [Ahyd / [Aald]). Ks is the dissociation constant (Ks =
([E][Aald]) / [EAald]) of the non-covalent chymotrypsin–aldehyde inhibitor
257
t2:1
t2:2
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.
a
Inhibitor
Ki(obs)
(μM)
KHK(obs)
KHA(obs)
KH1
Effective molarity
(M)
ΔG at 25 °C
(kJ/mol)
t2:3
(M−1
)
t2:4
t2:5
t2:6
t2:7
ZAAF-COCHO
ZAAF-CHO
0.37b
1.7
1200
0.028
0.25
41,000
6000
−26.3
−21.5
0.39
1500
KHA2
Diastereoisomer
KHA1
340
t2:8
t2:9
ZAAF-CHO
ZAAF-CHO
Hemiacetal-1
Hemiacetal-2
0.25
0.26
1400
4600
−17.9
−20.9
1100
a
t2:10
t2:11
Present work, errors are the standard deviations of 3 determinations.
[16].
b
Please cite this article as: J.A. Cleary, et al., Hemiacetal stabilization in a chymotrypsin inhibitor complex and thereactivity of the hydroxyl group of
the catalytic..., Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.03.008

J.A. Cleary et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
5
Table 3
t3:1
t3:2
t3:3
t3:4
Titration constants from pH dependent changes in the chemical shifts of the 13C-enriched
carbons of Z-Ala-Ala-Phe-13CHO and Z-Ala-Ala-Phe-13COCHO when they form complexes
with α-chymotrypsin.
Inhibitor
δ1
δ2
δ3
δ2–δ1 δ3–δ2 δ3–δ1 pKa1 pKa2
t3:5
(ppm) (ppm) (ppm) (ppm) (ppm) (ppm)
ZAAF-13CHOa
ZAAF-13CHOb
99.25 100.14
0.89
98.99 2.11
7.00
6.86 10.65
5.52
t3:6
t3:7
t3:8
95.55
97.66
1.33
0.32
3.44
3.53
ZAAF-13COCHOc 100.79 104.00 104.32 3.21
7.89
a
Present work, fitted values from Fig. 2C.
Present work, fitted values from Fig. 2B.
Fitted data from [16].
t3:9
b
t3:10
t3:11
c
change which inactivates chymotrypsin [19,24]. The binding of ligands 310
and or chemical modification of the active site serine hydroxyl is known 311
to raise the pKa of the isoleucine-16/aspartate-194 ion pair to a 312
value of 10.5–11.0 [16,19] and so we assign the pKa of 10.65 to ion- 313
ization of the isoleucine-16/aspartate-194 ion pair. The smaller sig- 314
nal at 99.2 ppm(Fig. 1c) titrated with a similar pKa of 7.0(Fig. 2C) 315
but the titration shift (δ2-δ1) was smaller (Table 3).
316
OOF
The pKa values of ~7.0 for the hemiacetal oxyanions in the inhibitor 318
3.6. Oxyanion stabilization
317
complexes are much smaller than the oxyanion pKa of 13.23 0.1 319
for the inhibitor hydrate (Fig. 2A). This shows that chymotrypsin is sta- 320
bilizing the hemiacetal oxyanion of both diastereoisomers by similar 321
amounts. In order to quantify this stabilization we need to determine 322
the hemiacetal pKa in the absence of enzyme stabilization. This can be 323
done using free energy relationships [25]. The pKa of ZAAF-13COOH 324
(Scheme 1B) was determined by 13C-NMR to be 3.52
0.03. Using 325
the relationship σ* = (4.644-pKa)/1.7 [26] for ZAAF-COOH the substit- 326
uent constant, σ* = 0.66 for the substituent ZAAF\. The substituent 327
constant for EO\ is 1.68 [27] and using these σ* values and the relation- 328
ship pKa = 15.9–1.42 Σσ* [28] we estimate that the pKa of the oxyanion 329
of the hemiacetal formed when chymotrypsin and Z-Ala-Ala-Phe-CHO 330
react will be 12.6. Therefore chymotrypsin has lowered the pKa of the 331
larger hemiacetal-1 (Fig. 1c) by 12.6–7.0 = 5.6 pKa units and the pKa 332
of the smaller hemiacetal-2 (Fig. 1c) by a similar amount (Table 4). 333
The oxyanions of both hemiacetals are therefore both stabilized by 334
~32 kJ mol−1 (Table 4).
335
3.7. Effect of pH on hemiacetal and hemiketal formation
336
With the glyoxal inhibitor (ZAAF-COCHO) there is a ~40 fold de- 337
crease in binding when the pH is decreased from 7.2 to 3.2 and there 338
is a smaller ~2.6 fold decrease in binding on increasing the pH from 339
7.2 to 10.6 (Table 5). However, with the aldehyde inhibitor (ZAAF- 340
CHO) there is a much smaller decrease in binding of ~3 when the pH 341
is decreased to from 7.2 to 3.2 (Table 5). However, from pH 7.2 to 10.6 342
there is an ~3 fold decrease in binding for both aldehydes and glyoxals 343
(Table 5). A similar small 4 fold increase in Ki from pH 8 to 3 with 344
Bz-Phe-CHO and chymotrypsin has led to the suggestion that the hemi- 345
acetal is neutral throughout the pH range 3–8 [11,15]. As our results 346
show that oxyanion formation occurs according to a pKa of ~7 then 347
we conclude that this suggestion is incorrect and the hemiacetal is not 348
neutral at pH 7 or higher. 349
With ZAAF-COCHO hemiketal formation (KHKobs) is unchanged from 350
pH 7.2 to 3.2 (Table 5) 351
Fig. 2. Effect of pH on the chemical shift of the hydrate and the hemiacetal 13C-NMR signals of
Z-Ala-Ala-Phe-13CHO observed in the presence of α-chymotrypsin. In (A) and (C) the contin-
uous lines were calculated using the equation δobs = δ1 / (1 + Ka/[H]) + δ2 / (1 + [H]/Ka)
and for the hydrate signal in (A) the fitted parameters were pKa = 13.23
0.01, δ1 =
91.04
0.01 ppm and δ2 = 95.28
0.24 ppm. For the hemiacetal signal in (C) the
fitted parameters were pKa = 7.00
0.16, δ1 = 99.25 0.02 ppm and δ2 = 100.14
UNCORRECTED PR
0.02. ppm. For the hemiacetal signal in (B) the continuous line was calculated using
the equation δobs = (δ1[H]2 + δ2Ka1[H] + Ka1Ka2Ka3)/([H]2 + Ka1[H]) + Ka1Ka2
and the fitted parameters pKa1 = 6.86 0.05, pKa2 = 10.65 0.02, δ1 = 95.55
0.02 ppm, δ2 = 97.66 0.02 ppm and δ3 = 98.99 0.04 ppm.
)
303 of 10.65 0.12 (Fig. 2B). The total titration shift of 3.44 ppm is similar to
304 that of 3.53 ppm observed for formation of the hemiketal oxyanion with
305 the corresponding hemiketal formed by the glyoxal inhibitor Z-Ala-Ala-
306 Phe-13COCHO reacting with chymotrypsin(Table 3). Therefore the
307 titration shift is assigned to formation of the hemiacetal oxyanion
However, as a result of the better binding of the aldehyde compared 352
to the glyoxal at pH 3.2 there is a ~2 fold increase in hemiacetal forma- 353
tion (KHAobs) from pH 7.2 to 3.2 with ZAAF-CHO (Table 5). In contrast 354
with ZAPF-COCHO hemiketal formation decreases from pH 7.2 to 3.2 355
or 10.2 [4].
356
308
when Z-Ala-Ala-Phe-CHO forms a hemiacetal with chymotrypsin. Ioniza-
tion of the isoleucine-16/aspartate-194 ion pair causes a conformational
The binding of ZAAF-H decreased 6 fold from pH 7.2 to 10.2 and both 357
aldehyde and glyoxal binding decreased ~3 fold from pH 7.2 to 10.2 358
309
Please cite this article as: J.A. Cleary, et al., Hemiacetal stabilization in a chymotrypsin inhibitor complex and thereactivity of the hydroxyl group of
the catalytic..., Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.03.008

6
J.A. Cleary et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
t4:1
t4:2
Table 4
Hemiacetal and hemiketal oxyanion stabilization in aldehyde (ZAAF-CHO) and glyoxal (ZAAF-COCHO and ZAPFCOCHO) inhibitor complexes formed with α-chymotrypsin.
t4:3
Complex formed
Inhibitor complexed with chymotrypsin
Oxyanion pKa
pKa decrease
Oxyanion stabilization
pKaw
(water)
pKae
(enzyme)
pKaw–pKae
ΔG
t4:4
(kJ mol−1
)
t4:5
t4:6
t4:7
Hemiacetal-1
Hemiacetal-2
Hemiketal
ZAAF-13CHO
ZAAF-13CHO
ZAAF13COCHO
12.6a
12.6a
9.5d
7.0b
6.9c
4.6e
5.6
5.7
4.9
32.0
32.5
28.0
a
t4:8
pKa of hemiacetal in water calculated as described in the text.
pKa of hemiacetal-1 formed with α-chymotrypsin as in Scheme 1.
pKa of hemiacetal-2 formed with α-chymotrypsin as in Scheme 1.
pKa 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].
pKa of hemiketal formed with chymotrypsin [16].
b
c
t4:9
t4:10
t4:11
t4:12
d
e
359 (Table 5). Similar decreases were observed with ZAPF-H and ZAPF-
360 COCHO [4] supporting the suggestion [16] that this increase is due to a
361 conformational change resulting from the ionization of the isoleucine-
362 16/aspartate-194 ion pair in chymotrypsin.
pKa values in free chymotrypsin and in the Z-Ala-Ala-Phe-CHO- 396
chymotrypsin complex were 7.32
(Fig. 5C) respectively at 4 °C. Similar values of 7.35
7.87
0.18 (Fig. 5D) and 7.97
0.20 397
0.09 and 398
0.18 were obtained for the free chymotrypsin (Fig. 5B) and 399
the Z-Ala-Ala-Phe-CHO–chymotrypsin complex (Fig. 5A) respec- 400
tively at 25 °C. The histidine pKa value for the free enzyme is similar 401
to the values of 6.7 and 7.5 reported for α- and δ-chymotrypsin re- 402
spectively [16,32]. The increase in the histidine pKa from 7.3 to 7.9 403
on adding Z-Ala-Ala-Phe-CHO to α-chymotrypsin contrasts with 404
the observed decrease in the histidine pKa to 6.5 when Acetyl-Leu- 405
363 3.8. 1H-NMR of the hydrogen bonded protons of α-chymotrypsin and of its
364 complex with Z-Ala-Ala-Phe-CHO
365
In chymotrypsin signals at ~18 and ~13 ppm have been assigned to
366 the Nδ1 and Nε2 protons of the positively charged imidazolium ring of
367 histidine-57 and the signal at ~15 ppm has been assigned to the Nδ1
368 proton of the neutral imidazole ring [16,29–32]. In the glyoxal complex
369 of α-chymotrypsin with Z-Ala-Ala-Phe-COCHO the signals at ~18 ppm
370 and ~13 ppm are present at both low and high pHs at 25 °C demonstrat-
371 ing that the binding of the glyoxal inhibitor has raised the pKa of the ac-
372 tive site histidine to a value N11 [16]. In contrast, in the aldehyde
373 complex of α-chymotrypsin and Z-Ala-Ala-Phe-CHO the signals were
374 barely discernable at 25 °C (Fig. 3A) and they were similar to those ob-
375 served from free α-chymotrypsin under the same conditions (Fig. 3B).
Phe-CHO binds to α-chymotrypsin [14].
406
4. Discussion
407
Aldehydes have been widely used as potent inhibitors of the serine 408
proteases [1–3,33] and our results show that the aldehyde group can in- 409
crease inhibitor potency by a factor of 100 (Table 1). The potency of this 410
inhibition is usually attributed to the ability of the aldehyde group to 411
react with active site serine hydroxyl group to form a hemiacetal that 412
mimics the tetrahedral intermediate formed during substrate catalysis 413
[6,15]. The fact that the aldehyde group of ZAAF-CHO is readily hydrated 414
(KH1obs = 13.9) illustrates how readily hydroxyl groups react with alde- 415
hyde carbonyl carbon to give a tetrahedral species. However, solvent 416
water hydroxyl groups compete with the enzymes catalytic serine hy- 417
droxyl decreasing the concentration of the non-hydrated aldehyde 418
group which reacts with hydroxyl group of the catalytic serine residue. 419
Therefore the observed Ki values are normally corrected to allow for this 420
using Kiobs = Ki(1 + KHYD). In our case the Ki value for ZAAF-CHO would 421
be decreased by a factor of 14.9 to 0.11 μM. For ZAAFCOCHO we have 422
shown that the active site serine hydroxyl group of chymotrypsin reacts 423
with the keto carbonyl of the glyoxal group which has a lower KH1obs = 424
1.58 therefore the observed Ki will only be reduced by a factor of 2.58 to 425
0.14 μM which is similar to the corrected Ki value of 0.11 μM obtained 426
with the aldehyde inhibitor. Therefore the fact that glyoxal inhibitors 427
are bound ~4 fold more tightly than the corresponding aldehyde inhib- 428
itor is due to the fact that with the glyoxal inhibitors hemiketal forma- 429
tion occurs at the less effectively hydrated keto carbon as opposed to 430
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
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 inhib-
itor 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 suppres-
sion 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
the more heavily hydrated aldehyde carbon.
431
Inhibition of chymotrypsin by glyoxal inhibitors has been shown to 432
be stereospecific with only one diastereomeric hemiketal being formed 433
[16,17]. However, with the corresponding peptide aldehyde inhibitor, 434
inhibition is not fully stereospecific with two diastereoisomers being 435
formed. The diastereoisomer at 95.5 ppm predominates (~75%) with 436
the diastereoisomer at 99.2 ppm making up the remaining ~25% of the 437
hemiacetal formed. Two diastereoisomers were also observed when 438
N-Acetyl-Phe-CHO was incubated with chymotrypsin but the proportions 439
and chemical shifts were not reported [12]. Likewise when Leupeptin was 440
incubated with trypsin two similar diastereoisomers were detected with 441
chemical shifts of 98.8 ppm (86%) and 97.2 ppm(14%) [13]. These titrated 442
with pKa values of 4.69 and 5.67. In the present work both the chymotryp- 443
sin diastereoisomers formed with Z-Ala-Ala-Phe-CHO titrated but their 444
t5:1
t5:2
t5:3
Table 5
The effect of pH on the formation of hemiacetals or hemiketals in chymotrypsin inhibitor
complexes.
t5:4
t5:5
pH
Ki(obs)a (μM)
KHA(obs)
KHK(obs)
ZAAF-H
ZAAF-CHO
ZAAF-COCHO
ZAAF-CHO
ZAAF-COCHO
t5:6
t5:7
t5:8
3.2
7.2
10.6
1100
170
1100
60
10
110
5.4
1.7
5.5
0.4
0.4
0.8
15.0b
0.37b
0.967b
3100
1500
2900
1200
1200
2800
a
t5:9
Present work, errors are the standard deviations of 3 determinations.
[16].
b
t5:10
Please cite this article as: J.A. Cleary, et al., Hemiacetal stabilization in a chymotrypsin inhibitor complex and thereactivity of the hydroxyl group of
the catalytic..., Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.03.008

J.A. Cleary et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
7
OOF
Fig. 3. pH dependence of the 1H-NMR spectra of α-chymotrypsin and its complex with Z-Ala-Ala-Phe-13CHO at 4 and 25 °C obtained using the Watergate-5 pulse sequence. 1H-NMR
acquisition and processing parameters were as described in the Materials and methods section. All samples contained 0.6-0.7% (v/v) d6-dimethyl sulphoxide, 9.4–10.0 mM potassium
phosphate and 10% (v/v) 2H2O which was used as a deuterium lock signal. Sample conditions were; (A) volumes 0.75–0.80 mL, 1.71–1.82 mM α-chymotrypsin and 1.76–1.88 mM
Z-Ala-Ala-Phe-CHO; (B) volumes 0.75–0.82 mL, 1.69–1.84 mM α-chymotrypsin and 1.72–1.88 mM Z-Ala-Ala-Phe-CHO; (C) volumes 0.75–0.78 mL, 1.76–1.83 mM α-chymotrypsin
and 1.81–1.88 mM Z-Ala-Ala-Phe-CHO;(D) volumes 0.75–0.78 mL, 1.76–1.83 mM α-chymotrypsin and 1.81–1.88 mM Z-Ala-Ala-Phe-CHO.
445
446
447
pKa values were very similar at 6.9 and 7.0 (Table 4) suggesting that they
are both in similar environments.
from the backbone amide nitrogens of serine-195 and glycine-193. 454
However, in the other diastereoisomer the oxyanion is about 2.9 Å 455
from the Nε2 nitrogen of the positively charged imidazolium ion of 456
histidine-57. Therefore the oxyanions are in different electrostatic envi- 457
ronments in the two diastereoisomers and so we would expect that 458
they would have significantly different pKa values as is observed for 459
the two diastereoisomeric hemiacetals observed in the Acetyl-Leu- 460
Phe-CHO/trypsin complex [14]. The fact that in the Z-Ala-Ala-Phe- 461
CHO/chymotrypsin complex the oxyanion pKa values were ~7 for both 462
X-ray crystallographic studies of aldehyde inhibitors binding to
448 serine proteases [14,34,35] has shown that hemiacetal formation is
449 not stereospecific with aldehyde inhibitors with two diastereomeric
450 hemiacetals being formed. One of these has the oxyanion in the
451 oxyanion hole while in the other diastereoisomer the oxyanion points
452 towards the active site histidine [14,34,35]and is hydrogen bonded to
453 Nε2 of histidine-57. The oxyanion in the oxyanion hole is about 2.8 Å
UNCORRECTED PR
Fig. 4. pH dependence of the 1H-NMR spectra of α-chymotrypsin and its complex with Z-Ala-Ala-Phe-13CHO at 4 and 25 °C obtained using the 1331-pulse sequence. 1H-NMR acquisition
and processing parameters were as described in the Materials and methods section. All samples contained 0.6–0.7% (v/v) d6-dimethyl sulphoxide and 10% (v/v) 2H2O which was used as a
deuterium lock signal. Sample conditions were; (A) volumes 0.75–0.79 mL, 1.76–1.84 mM α-chymotrypsin, 1.80–1.88 mM Z-Ala-Ala-Phe-CHO and 9.5–10.0 mM potassium phosphate;
(B) volumes 0.75–0.82 mL, 1.72–1.84 mM α-chymotrypsin, 1.74–1.88 mM Z-Ala-Ala-Phe-CHO and 9.3–10.0 mM potassium phosphate; (C) and (D) volumes 0.75–0.80 mL, 1.72–1.83 mM
α-chymotrypsin, 1.81–1.88 mM Z-Ala-Ala-Phe-CHO and 9.4–10.0 mM potassium phosphate.
Please cite this article as: J.A. Cleary, et al., Hemiacetal stabilization in a chymotrypsin inhibitor complex and thereactivity of the hydroxyl group of
the catalytic..., Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.03.008

8
J.A. Cleary et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
Table 6
t6:1
t6:2
t6:3
t6:4
Titration constants from pH dependent changes in the chemical shift of the Nδ1 proton of
histidine-57 in α-chymotrypsin and in the Z-Ala-Ala-Phe-CHO/α-chymotrypsin complex
at 4 and 25 °C.
Inhibitor
°C
δ1
δ2
δ2–δ1
pKa
t6:5
(ppm)
(ppm)
(ppm)
ZAAF-13CHO
None
25
25
4
17.85
18.11
17.79
18.01
13.84
13.73
14.54
14.56
3.01
3.38
3.25
4.45
7.87
7.35
7.97
7.32
t6:6
t6:7
t6:8
t6:9
ZAAF-13CHO
None
4
diastereoisomers suggests that the oxyanions in both diastereoisomers 463
are in similar electrostatic environments. 464
Since the binding Z-Ala-Ala-Phe-CHO raised the pKa of histidine-57 465
to ~8 (Fig. 5) and the oxyanion pKa values of the diastereomeric hemi- 466
acetals were both ~7 (Fig. 2) it is expected that the positively charged 467
histidine residue will play an important role in lowering the oxyanion 468
pKa to ~7. The binding of Acetyl-Leu-Phe-CHO to α-chymotrypsin 469
resulted in the formation of a hemiacetal signal at 102 ppm which did 470
not titrate from pH 7 to 13 [14]. The chemical shift value of 102 ppm 471
is larger than that observed (~100 ppm) when the oxyanion with 472
Z-Ala-Ala-Phe-CHO is fully formed (Fig. 2B,C). This suggests that the 473
oxyanion is fully formed at pH 7 with Acetyl-Leu-Phe-CHO and so 474
should have a pKa b 6. The histidine pKa of 6.5 in the Acetyl-Leu-Phe- 475
CHO complex [14] shows that it should be able to stabilize the oxyanion. 476
However, with glyoxal inhibitors the histidine pKa is raised to N 11 477
therefore it is clear that while aldehyde inhibitors like glyoxal inhibitors 478
are bound tightly to chymotrypsin, they are much less effective at 479
raising the pKa of the active site histidine in chymotrypsin. Although 480
oxyanion stabilization is more effective with the aldehyde hemiacetal 481
oxyanion than with the glyoxal hemiketal oxyanion (Table 4) the pKa 482
of the imidazolium ion of histidine-57 is only raised to ~8 with hemiac- 483
etal formation (Table 6) while it is raised to N11 with hemiketal forma- 484
tion in glyoxal complexes [16] inhibitor and to N10 when peptidyl 485
trifluoromethyl ketones [36] or peptidyl boronic acid [37] inhibitors 486
form negatively charged tetrahedral adducts with chymotrypsin. This 487
shows that oxyanion formation only has a minor role in raising the 488
pKa of the imidazolium ion of histidine-57 in aldehyde inhibitor com- 489
plexes. Therefore replacing the aldehyde hydrogen with an aldehyde 490
group [16,17] or trifluoromethylene group [38] is the major factor in 491
strengthening the hydrogen bond between histidine-57 and aspartate- 492
102, raising the pKa of the imidazolium ion of histidine-57 and ensuring 493
that tetrahedral intermediate formation is stereospecific. Similar effects 494
are expected during catalysis with substrates.
495
Therefore tetrahedral adduct formation only increases the strength 496
of the hydrogen bond between the aspartate and histidine residues rais- 497
ing the pKa of the imidazolium ion of histidine-57 to a value N 10 if the 498
leaving group peptide nitrogen is present or if an inhibitor carbon atom 499
is present in an equivalent position. This suggests that the inhibitor 500
aldehyde hydrogen atom is not large enough to compress histidine- 501
57-aspartate-32 hydrogen bond and/or screen it from solvent so that 502
the histidine pKa is raised to a value N10. Likewise a the aldehyde proton 503
must be replaced by a larger group to ensure that the formation of the 504
tetrahedral species is stereospecific.
505
The catalytic serine group is expected to have a pKa of ~15 and so for 506
the imidazolium ion of histidine-57 to act as an effective base catalyst its 507
pKa should be raised from its value of 7 in the free enzyme to a value 508
similar to that of the active site serine hydroxyl group when a substrate 509
Fig. 5. Effect of pH on the chemical shift of the Nδ1 hydrogen bonded proton of histidine-57
in α-chymotrypsin and its complex with Z-Ala-Ala-Phe-13CHO at 4 and 25 °C. In (A),
(B), (C) and (D) the continuous lines were calculated using the equation δobs = δ1 / (1 +
K
a / [H]) + δ2 / (1 + [H]/Ka) and the fitted parameters were: (A) pKa = 7.87 0.18, δ1
17.85 0.06 ppm and δ2 = 14.84 0.06 ppm, (B) pKa = 7.35 0.09, δ1 = 18.11
0.05 ppm and δ2 = 14.73 0.04. ppm, (C) pKa = 7.97 0.20, δ1 = 17.79 0.08 ppm
0.18, δ1 = 18.01 0.12 ppm
=
and δ2 = 14.54
and δ2 = 14.56
0.08 ppm and (D) pKa = 7.32
0.09 ppm.
Please cite this article as: J.A. Cleary, et al., Hemiacetal stabilization in a chymotrypsin inhibitor complex and thereactivity of the hydroxyl group of
the catalytic..., Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.03.008

J.A. Cleary et al. / Biochimica et Biophysica Acta xxx (2014) xxx–xxx
[9] P. Wyeth, R.P. Sharma, M. Akhtar,
9
A
proton-magnetic-resonance study of 575
510 binds [4,22]. If the aldehyde inhibitor has only a small effect on the pKa
511 of the active site histidine before tetrahedral adduct formation, then the
512 fact that the effective molarity of the hydroxyl group of the catalytic
513 serine group is only ~7× lower with the aldehyde inhibitor compared
514 with its equivalent glyoxal inhibitor (Table 2) shows that a large in-
515 crease in the pKa of histidine-57 is not required to increase the reactivity
516 of the serine hydroxyl group for formation of the tetrahedral hemiace-
517 tal. This suggests that the expected increase in the effective molarity
518 of the active site serine hydroxyl required for tetrahedral intermediate
519 formation during catalysis cannot be solely attributed to a large increase
520 in the pKa of histidine-57. Therefore we propose that the primary reason
521 for the increase in the effective molarity of the catalytic hydroxyl when
522 it binds substrates will be the entropic advantage achieved by optimally
523 aligning the catalytic serine's hydroxy group with the carbonyl carbon
524 of the peptide being hydrolysed.
N-trifluoroacetyl-L-alanyl-L-phenylalaninal binding to alpha-chymotrypsin, Eur. J. 576
Biochem. 105 (1980) 581–585. 577
[10] D.G. Gorenstein, D.O. Shah, Proton and fluorine nuclear magnetic resonance spectro- 578
scopic observation of hemiacetal formation between N-acyl-p-fluorophenylalaninals 579
and alpha-chymotrypsin, Biochemistry 21 (1982) 4679–4686.
580
[11] D.O. Shah, D.G. Gorenstein, Fluorine nuclear magnetic resonance spectroscopy of 581
“transition state analogue” complexes of the D and L enantiomers of N-Acetyl-p- 582
fluorophenylalinal and alpha-chymotrypsin, Biochemistry 22 (1983) 6096–6101. 583
[12] D.O. Shah, K. Lai, D.G. Gorenstein, 13C NMR spectroscopy of “transition-state ana- 584
logue” complexes of N-acetyl-L-phenylalaninal and alpha-chymotrypsin, J. Am. 585
Chem. Soc. 106 (1984) 4272–4273.
586
[13] C. Ortiz, C. Tellier, H. Williams, N.J. Stolowich, A.I. Scott, Diastereotopic covalent binding 587
of the natural inhibitor leupeptin to trypsin: detection of two interconverting 588
hemiacetals by solution and solid-state NMR spectroscopy, Biochemistry 30 589
(1991) 10026–10034.
590
[14] D. Neidhart, Y. Wei, C. Cassidy, J. Lin, W.W. Cleland, P.A. Frey, Correlation of 591
low-barrier hydrogen bonding and oxyanion binding in transition state analogue 592
complexes of chymotrypsin, Biochemistry 40 (2001) 2439–2447.
593
[15] W.P. Kennedy, R.M. Schultz, Mechanism of association of a specific aldehyde “tran- 594
sition-state analogue” to the active site of alpha-chymotrypsin, Biochemistry 18 595
525
We conclude that a moiety larger than the aldehydic proton must be
(1979) 349–356.
596
[16] E. Spink, S. Cosgrove, L. Rogers, C. Hewage, J.P.G. Malthouse, 13C and 1H NMR studies 597
of ionizations and hydrogen bonding in chymotrypsin-glyoxal inhibitor complexes, 598
526 present if the pKa of the active site histidine is to be raised to a value N10
527 and if there is to be stereospecific formation of a tetrahedral adduct.
528 With aldehyde inhibitors there is stabilization of the hemiacetal
529 oxyanion with its pKa being reduced by ~5.6 pKa units while the pKa
530 of the active site histidine is only raised ~0.5 pKa units. Therefore we
531 conclude that a large increase in the pKa of the active site histidine is
532 not required for oxyanion stabilization or for the active site serine
533 hydroxyl group to have an effective molarity of 6000. As the active site
534 serine hydroxyl group is expected to have a pKa ~15 then we have ar-
535 gued [4,16] that the large increase in the pKa of the active site histidine
536 (i.e. pKa N 11) observed on binding glyoxal inhibitor is required for it to
537 increase the reactivity of the serine hydroxyl group by general acid ca-
538 talysis. But, with the aldehyde inhibitor there is only a small increase
539 in the pKa of the active site histidine pKa to ~8. However, the effective
540 molarity of the serine hydroxyl group with the aldehyde inhibitor is
541 only ~7 fold smaller than that observed with the corresponding glyoxal
542 inhibitor (Table 2). This clearly demonstrates that only a small part of
543 the high reactivity of the serine hydroxyl group can be attributed to gen-
544 eral base catalysis by the catalytic histidine residue of chymotrypsin.
J. Biol. Chem. 282 (2007) 7852–7861.
599
[17] A. Djurdjevic-Pahl, C. Hewage, J.P.G. Malthouse, A 13C-NMR study of the inhibition of 600
delta-chymotrypsin by a tripeptide-glyoxal inhibitor, Biochem. J. 362 (2002) 339–347. 601
[18] S. Cosgrove, L. Rogers, C. Hewage, J.P.G. Malthouse, An NMR study of the inhibition 602
OOF
of pepsin by glyoxal inhibitors: mechanism of tetrahedral intermediate stabilization 603
by the aspartyl proteinases, Biochemistry 46 (2007) 11205–11215.
604
[19] M.D. Finucane, E.A. Hudson, J.P.G. Malthouse, A 13C-N.M.R. investigation of the ion- 605
izations within an inhibitor-alpha-chymotrypsin complex: evidence that both 606
alpha-chymotrypsin and trypsin stabilize a hemiketal oxyanion by similar mecha- 607
nisms, Biochem. J. 258 (1989) 853–859.
608
[20] M. Liu, X. Mao, C. He, H. Huang, J.K. Nicholson, J.C. Lindon, Improved WATERGATE 609
pulse sequences for solvent suppression in NMR spectroscopy, J. Magn. Reson. 132 610
(1998) 125–129.
[21] P.J. Hore, Solvent suppression in Fourier transform nuclear magnetic resonance, J. 612
Magn. Reson. 55 (1983) 283–300. 613
[22] M.D. Finucane, J.P.G. Malthouse, A study of the stabilization of tetrahedral adducts 614
by trypsin and delta-chymotrypsin, Biochem. J. 286 (1992) 889–900. 615
611
[23] M.P. Gamcsik, J.P.G. Malthouse, W.U. Primrose, N.E. Mackenzie, A.S.F. Boyd, R.A. 616
Russell, A.I. Scott, Structure and stereochemistry of tetrahedral inhibitor complexes 617
of papain by direct NMR observation, J. Am. Chem. Soc. 105 (1983) 6324–6325.
618
[24] A.R. Fersht, Y. Requena, Equilibrium and rate constants for the interconversion of 619
two conformations of α-chymotrypsin. The existence of a catalytically inactive con- 620
formation at neutral pH, J. Mol. Biol. 60 (1971) 279–290.
[25] D.D. Perrin, B. Dempsey, E.P. Serjeant, pKa Prediction for Organic Acids and Bases, 622
Chapman and Hall, London and New York, 1981. 623
621
545 Acknowledgements
[26] S. Takahashi, L.A. Cohen, H.K. Miller, E.G. Peake, Calculation of the pKa values of al- 624
cohols from σ* Constants and from the carbonyl frequencies of their esters, J. Org. 625
Chem. 36 (1971) 1205–1209.
[27] N. Howe, L. Rogers, C. Hewage, J.P.G. Malthouse, Oxyanion and tetrahedral interme- 627
diate stabilization by subtilisin: detection of a new tetrahedral adduct, Biochim. 628
626
546
This work was supported by the Irish Research Council and Uni-
547 versity College Dublin. Grant nos. 055637/Z/98 from the Wellcome
548 Trust was used to purchase the NMR spectrometer used in these
549 studies and Science Foundation Ireland funding was used to upgrade
550 the NMR spectrometer.
Biophys. Acta, Proteins Proteomics 1794 (2009) 1251–1258.
629
[28] P. Ballinger, F.A. Long, Acid ionization constants of alcohols. II. Acidities of some 630
substituted methanols and related compounds, J. Am. Chem. Soc. 82 (1960) 795–798. 631
[29] W.W. Bachovchin, Confirmation of the assignment of the low-field proton reso- 632
nance of serine proteases by using specifically nitrogen-15 labeled enzyme, Proc. 633
Natl. Acad. Sci. U. S. A. 82 (1985) 7948–7951.
634
551 References
[30] J.L. Markley, W.M. Westler, Protonation-state dependence of hydrogen bond 635
strengths and exchange rates in a serine protease catalytic triad: bovine chymotryp- 636
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
[1] A.M. LeBeau, P. Singh, J.T. Isaacs, S.R. Denmeade, Prostate-specific antigen is a
“chymotrypsin-like” serine protease with unique P1 substrate specificity, Biochem-
istry 48 (2009) 3490–3496.
[2] M. Mroczkiewicz, K. Winkler, D. Nowis, G. Placha, J. Golab, R. Ostaszewski, Studies of
the synthesis of all stereoisomers of MG-132 proteasome inhibitors in the tumor
targeting approach, J. Med. Chem. 53 (2010) 1509–1518.
sinogen A, Biochemistry 35 (1996) 11092–11097.
[31] G. Robillard, R.G. Shulman, High resolution nuclear magnetic resonance study of the 638
histidine-aspartate hydrogen bond in chymotrypsin and chymotrypsinogen, J. Mol. 639
Biol. 71 (1972) 507–511.
[32] G. Robillard, R.G. Shulman, High resolution nuclear magnetic resonance studies of 641
the active site of chymotrypsin, J. Mol. Biol. 86 (1974) 519–540. 642
[33] R.L. Stein, A.M. Strimpler, Slow-binding inhibition of chymotrypsin and cathepsin G 643
by the peptide aldehyde chymostatin, Biochemistry 26 (1987) 2611–2615. 644
637
640
[3] K. Akaji, H. Konno, H. Mitsui, K. Teruya, Y. Shimamoto, Y. Hattori, T. Ozaki, M.
Kusunoki, A. Sanjoh, Structure-based design, synthesis, and evaluation of peptide-
mimetic SARS 3CL protease inhibitors, J. Med. Chem. 54 (2011) 7962–7973.
[34] L.T.J. Delbaere, G.D. Brayer, The 1.8 Å structure of the complex between chymostatin 645
and Streptomyces griseus protease A. A model for serine protease catalytic tetrahe- 646
UNCORRECTED PR
[4] T. Petrillo, C.A. O'Donohoe, N. Howe, J.P.G. Malthouse, Importance of tetrahedral
intermediate formation in the catalytic mechanism of the serine proteases chymo-
trypsin and subtilisin, Biochemistry 51 (2012) 6164–6170.
dral intermediates, J. Mol. Biol. 183 (1985) 89–103.
[35] I.V. Kurinov, R.W. Harrison, Two crystal structures of the leupeptin–trypsin complex, 648
Protein Sci. 5 (1996) 752–758. 649
[36] T.C. Liang, R.H. Abeles, Inhibition of papain by nitriles: mechanistic studies using 650
NMR and kinetic measurements, Arch. Biochem. Biophys. 252 (1987) 626–634. 651
[37] S. Zhong, K. Haghoo, C. Kettner, F. Jordan, Proton magnetic resonance studies of 652
the active center histidine of chymotrypsin complexed to peptideboronic acids: 653
solvent accessibility to the N-delta and N-epsilon sites can differentiate 654
slow-binding and rapidly reversible inhibitors, J. Am. Chem. Soc. 117 (1995) 655
647
[5] J.O. Westerik, R. Wolfenden, Aldehydes as inhibitors of papain, J. Biol. Chem. 247
(1972) 8195–8197.
[6] R.C. Thompson, Use of peptide aldehydes to generate transition-state analogs of
elastase, Biochemistry 12 (1973) 47–51.
[7] R. Chen, D.G. Gorenstein, W.P. Kennedy, G. Lowe, D. Nurse, R.M. Schultz, Evidence for
hemiacetal formation between N-acyl-L-phenylalaninals and alpha-chymotrypsin by
cross-saturation nuclear magnetic resonance spectroscopy, Biochemistry 18 (1979)
921–926.
[8] G. Lowe, D. Nurse, Evidence for hemiacetal formation between alpha-chymotrypsin
and hydrocinnamaldehyde by cross-saturation nuclear magnetic resonance
spectroscopy, J. Chem. Soc. Chem. Commun. (1977) 815–816.
7048–7055.
656
[38] T.C. Liang, R.H. Abeles, Complex of α-chymotrypsin and N-acetyl-L-leucyl- 657
L-phenylalanyl trifluoromethyl ketone: structural studies with NMR spectroscopy, 658
Biochemistry 26 (1987) 7603–7608
659
660
Please cite this article as: J.A. Cleary, et al., Hemiacetal stabilization in a chymotrypsin inhibitor complex and thereactivity of the hydroxyl group of
the catalytic..., Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbapap.2014.03.008