Catalysis of hydrolysis and transesterification reactions of p- Nitrophenyl esters by a designed helix-loop-helix dimer

Article abstract of DOI:10.1021/ja970854s

KO-42, a polypeptide with 42 amino acid residues has been designed to fold into a hairpin helix-loop-helix motif that dimerizes and forms a four- helix bundle. The solution structure of the folded KO-42 dimer has, been determined by NMR and CD spectroscop

Full text of DOI:10.1021/ja970854s

11362
J. Am. Chem. Soc. 1997, 119, 11362-11372
Catalysis of Hydrolysis and Transesterification Reactions of
p-Nitrophenyl Esters by a Designed Helix-Loop-Helix Dimer
Kerstin S. Broo, Lars Brive, Per Ahlberg, and Lars Baltzer*
Contribution from the Department of Organic Chemistry, G o¨ teborg UniVersity,
4
12 96 G o¨ teborg, Sweden
X
ReceiVed March 17, 1997
Abstract: KO-42, a polypeptide with 42 amino acid residues has been designed to fold into a hairpin helix-loop-
helix motif that dimerizes and forms a four-helix bundle. The solution structure of the folded KO-42 dimer has
been determined by NMR and CD spectroscopy and ultracentrifugation. On the surface of the folded polypeptide
a reactive site has been engineered that is capable of catalyzing acyl-transfer reactions of reactive esters. The reactive
site of KO-42 contains six histidine residues with perturbed pKa values. The pKas of His-15, His-30, and His-34 are
close to 5, whereas those of His-11, His-19, and His-26 are close to 7, with nonideal titration curves. The second-
order rate constant for the KO-42 catalyzed hydrolysis of mono-p-nitrophenyl fumarate at pH 4.1 and 290 K is 0.1
-
1
1
-1
-5
M
M
s , which is 1140 times larger than that of the 4-methylimidazole (4-MeIm) catalyzed reaction, 8.8 × 10
-
-1
s . The second-order rate constant for the KO-42 catalyzed transesterification of mono-p-nitrophenyl fumarate
-
1
-1
to form the corresponding trifluoroethyl ester in 10 vol % trifluoroethanol at pH 4.1 and 290 K is 0.052 M
s
-
5
-1 -1
which is 620 times larger than that of the 4-MeIm catalyzed reaction, 8.4 × 10
M
s . KO-42 catalyzes the
corresponding reactions of other p-nitrophenyl esters with similar rate enhancements. At pH 4.1 in aqueous solution
3
where the rate constant ratio k2(KO-42)/k2(4-MeIm) is larger than 10 the predominant reactive species of KO-42
have unprotonated histidines flanked by protonated histidines. The kinetic solvent isotope effect at pH 4.7 is 2.0
which shows that isotopic fractionation occurs in the transition state. The kinetic solvent isotope effect at pH 6.1 is
1.1 which shows that there is neither general acid-general base catalysis nor strong hydrogen bonding in the transition
state of the rate-limiting reaction step at that pH. The results suggest that at low pH the dominant catalytic species
functions through a mechanism where unprotonated nucleophilic histidines are flanked by protonated histidines that
bind to one or both of the ester oxygens in the transition state.
Introduction
Designed polypeptide catalysts have been reported
1
0-12
previously,
but it is only in a few cases where the
De noVo designed proteins1,2 have great potential in the
engineering of novel catalysts because natural as well as non-
natural amino acids can be organized in three-dimensional space
to form complex reactive sites if the structure of the folded
peptide can be predicted. Designed four-helix bundles,
â-sandwich proteins, and a ââR motif have been reported to
fold in solution, and the formation of well-defined tertiary
relationship between structure and reactivity is at least partly
1
1
understood. Oxaldie, a peptide with 14 amino acid residues
that aggregates and forms amphiphilic helices, was reported to
catalyze the decarboxylation of oxaloacetate with a second-order
rate constant that was almost three orders of magnitude larger
than that of the ethyl amine catalyzed reaction. The secondary
structure was identified by NMR and CD spectroscopy, but the
tertiary structure could not be determined due to the dynamic
nature of the helical aggregate. A designed four-helix bundle
1
,3-6
7
8
structures has recently been demonstrated by NMR and CD
spectroscopy.3
,5,8
The introduction of reactive sites on the
surface of folded polypeptides is therefore timely, and the
engineering of hydrophobic cavities can now be envisioned
using, e.g., novel reactions for the posttranslational modification
of proteins.9
1
2
with a chymotrypsin-like active site was reported to catalyze
the hydrolysis of p-nitrophenyl acetate with a substantial rate
enhancement, but the active site was later found not to function
1
3
as intended.
We recently reported on the reactivity of functionalized
X
Abstract published in AdVance ACS Abstracts, November 1, 1997.
1) Bryson, J. W.; Betz, S. F.; Lu, H. S.; Suich, D. J.; Zhou, H. X.;
O'Neill, K. T.; DeGrado, W. F. Science 1995, 270, 935.
2) Mutter, M.; Vuilleumier, S. Angew. Chem., Int. Ed. Engl. 1989, 28,
35.
9
,14,15
(
helix-loop-helix dimers
where the reactive sites were
based on histidines flanked by residues capable of transition
state binding. The reactive polypeptides were developed from
(
5
1
2
1
(3) Raleigh, D. P.; Betz, S. F.; DeGrado, W. F. J. Am. Chem. Soc. 1995,
SA-42, a designed polypeptide that folded into a hairpin helix-
17, 7558.
loop-helix motif and dimerized to form a four-helix bundle.4
(4) Olofsson, S.; Johansson, G.; Baltzer, L. J. Chem. Soc., Perkin Trans.
The solution structure was determined by NMR and CD
1995, 2047.
(
5) Dolphin, G. T.; Brive, L.; Baltzer, L. J. Am. Chem. Soc. 1996. 118,
1247.
6) Fezoui, Y.; Weaver, D. L.; Osterhout, J. J. Proc. Natl. Acad. Sci.
U.S.A. 1994, 91, 3675.
7) Quinn, T. P.; Tweedy, N. B.; Williams, R. W.; Richardson, J. S.;
Richardson, D. C. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 8747.
spectroscopy and ultracentrifugation, and SA-42 was found to
4,16
form the designed motif.
The solution structures of the
(
(12) Hahn, K. W.; Klis, W. A.; Stewart, J. M. Science 1990, 248, 1544.
(13) Corey, M. J.; Hallakova, E.; Pugh, K.; Stewart, J. M. Appl. Biochem.
Biotechnol., 1994, 47, 199.
(
(
(
8) Struthers, M. D.; Cheng, R. P.; Imperiali, B. Science 1996,271, 342.
9) Broo, K.; Brive, L.; Lundh, A.-C.; Ahlberg, P.; Baltzer, L. J. Am.
(14) Baltzer, L.; Lundh, A.-C.; Broo, K.; Olofsson, S.; Ahlberg, P. J.
Chem. Soc., Perkin Trans. 2 1996, 1671.
Chem. Soc. 1996, 118, 8172.
(15) Broo, K.; Allert, M.; Andersson, L.; Erlandsson, P.; Stenhagen, G.;
Wigstr o¨ m, J.; Ahlberg, P.; Baltzer, L. J. Chem. Soc., Perkin Trans. 2 1997,
397.
(10) Gutte, B.; Daeumigen, M.; Wittschieber, E. Nature 1979, 281, 650.
11) Johnsson, K.; Allemann, R. K.; Widmer, H.; Benner, S. A. Nature
(
1
993, 365, 530.
(16) Olofsson, S.; Baltzer, L. Folding Design 1996. 1, 347.
S0002-7863(97)00854-8 CCC: $14.00 © 1997 American Chemical Society

Hydrolysis/Transesterification Reactions of p-Nitrophenyl Esters
J. Am. Chem. Soc., Vol. 119, No. 47, 1997 11363
Figure 1. The amino acid sequence of KO-42 in the one-letter code
where A is alanine, D is aspartic acid, E is glutamic acid, F is
phenylalanine, G is glycine, H is histidine, I is isoleucine, K is lysine,
L is leucine, N is asparagine, P is proline, Q is glutamine, R is arginine,
and V is valine. Aib is R-aminoisobutyric acid and Nle is norleucine.
The C-terminal is amidated and the N-terminal is acetylated. The
sequence is presented to illustrate the solution structure of the hairpin
motif.
polypeptides developed from SA-42 were determined by the
same techniques, and they, too, folded into helix-loop-helix
motifs. Flanking arginine, lysine, and ornithine residues were
found to enhance the reactivity of histidine side chains by factors
of 5-35, depending on the nature of the flanking side chain
and the solvent.17 Although the rate enhancements were modest,
the concept was successful, and we have now explored the use
of flanking protonated histidine side chains. In this Article we
wish to report that reactive sites with unprotonated histidine
side chains flanked by protonated histidine side chains are
capable of catalyzing acyl-transfer reactions of reactive esters
with second-order rate constants that are more than three orders
of magnitude larger than those of 4-methylimidazole. These
rate enhancements are the largest so far reported for rationally
designed four-helix bundles and pave the way for further
development of reactive sites in designed polypeptides capable
of substrate recognition and chiral discrimination.
Figure 2. Modeled structure of the hairpin helix-loop-helix motif
of KO-42 showing only the side chains of the histidine residues in the
a
reactive site. The measured pK values are given next to the residues
to which they have been assigned. KO-42 is a symmetric dimer, but
only the monomer is shown for reasons of clarity of presentation.
Results
The Design of KO-42. The design of KO-42 was based on
the design and solution structure of the template polypeptide
SA-42 which folds into a hairpin helix-loop-helix motif that
4
,16
dimerizes in an antiparallel mode to form a four-helix bundle.
4
The design of SA-42 has been described in detail, previously.
The amino acid sequence of KO-42 (Figure 1) is identical to
that of SA-42 except for five residues, His-11, His-19, His-26,
His-30, and His-34. The amino acid sequence of KO-42 is
Figure 3. The absolute value of the mean residue ellipticity of KO-
1
varied extensively to allow the assignment of the H NMR
4
2 at 222 nm as a function of pH at ambient temperature. The
spectrum, and two phenylalanine residues were incorporated at
the lower end of helix II to act as NOE reporter groups and to
generate increased shift dispersion in the folded peptide. The
choice of amino acids was governed to some extent by the
propensity for secondary structure formation,18 and some
artificial amino acids were used.
In the modeled polypeptide the folded states of the am-
phiphilic helical sequences were stabilized by capping residues,
salt bridges, and helix dipole stabilizing residues in agreement
with the generally accepted strategy that was recently reviewed
by Bryson et al.1 The C- and N-terminal charges were
neutralized by amidation and acylation, respectively. Leucine,
isoleucine, norleucine, and phenylalanine residues were incor-
porated to form shape complementary interfaces between the
helices.
concentration of peptide was 0.22 mM.
protonated histidine residues capable of binding one or both of
the ester oxygens in the transition state (Figure 2). Positively
charged hydrogen bond donors such as Arg, Lys, and Orn have
previously been found to provide only modest rate enhance-
ments.
k2(polypeptide)/k2(4-MeIm), for the reaction with mono-p-
nitrophenyl fumarate was typically less than 10 in aqueous
solution. In KO-42 flanking histidines were used instead.
9,14
The ratio of the second-order rate constants,
The Solution Structure of KO-42. The CD spectrum of
1
9
KO-42 is typical of helical proteins with minima at 208 and
2
(
2
other reported four-helix bundles and may be used to estimate
a helical content of more than 65%.
strongly dependent on pH (Figure 3), and at pH 2.5 the mean
22 nm. The mean residue ellipticity at 222 nm was -24 000
2
-1
1000 deg cm dmol in aqueous solution at pH 6.15 and
93 K. The measured ellipticity compares well with that of
On the surface of the folded polypeptide a reactive site was
engineered that was designed to catalyze acyl-transfer reactions
of esters using the catalytic efficiency of imidazole residues.
The designed reactive site contained unprotonated His residues
designed to function as nucleophilic catalysts flanked by
2
0,21
The CD spectrum is
2
-1
residue ellipticity is only -5000 deg cm dmol . The effect
of temperature on the CD spectrum is weak, the mean residue
(
17) Lundh, A.-C.; Broo, K.; Baltzer, L. J. Chem. Soc., Perkin Trans. 2
997, 209.
18) Richardson, J. S; Richardson, D. C. Prediction of Protein Structure
1
(19) Johnson, W. C., Jr. Proteins 1990, 7, 205.
(20) Chen, Y.-H.; Yang, J. T.; Chau, K. H. Biochemistry 1974, 13, 3350.
(21) Engel, M.; Williams, R. W.; Erickson, B. W. Biochemistry 1991,
30, 3161.
(
and the Principles of Protein Conformation; Fasman, G. D., Ed.; Plenum
Press: New York, 1989; pp 1-88.

11364 J. Am. Chem. Soc., Vol. 119, No. 47, 1997
Broo et al.
1
Figure 4. Summary of R proton chemical shifts and interresidue sequential and medium-range NOEs observed in the H NMR spectrum of KO-42
in 6 vol % TFE at pH 6.1 and 323 K. “Downward” bars indicate negative deviations of R proton chemical shifts relative to those of random coils
which is typical of residues in helical conformations. No bar is given for Aib residues since they have no R protons, and bars for Asp-23 and Ala-25
indicate that they have not been assigned. Solid lines indicate observed NOE contacts between residues, dashed line indicate possible NOE contacts
that are unobservable due to overlap, and solid thin lines are drawn for all possible contacts involving residues that have not been assigned.
3
2
Nomenclature is according to Wutrich.
ellipticity at 323 K is only 15% less than that at 293 K and
KO-42 does not have a well-defined melting point. Trifluoro-
ethanol (TFE) increases the absolute value of the mean residue
ellipticity in aqueous solution at pH 5.0, and most of the increase
takes place in the interval from 0 to 16 vol %. The increase is
small, less than 10%, which shows that the secondary structure
is well developed already in aqueous solution.
1
The H NMR spectrum of KO-42 has been assigned from
1
the 500 MHz H NMR TOCSY and NOESY spectra in 2, 4,
and 6 vol % of trifluoroethanol in 90% H2O 10% D2O at pH
5
.8 and 323 K according to the strategy developed for the
4,16
structural characterization of SA-42.
Small amounts of TFE
sharpen NMR resonances and simplify assignments but do not
induce tertiary structure.16 The chemical shifts of the R protons
in 6 vol % (1.5 mol %) TFE in the sequences 3-16 and 27-
4
2 show negative deviations relative to those of random coils,
22
which is indicative of helical structure (Figure 4). The helical
regions correspond closely to those determined for the template
polypeptide SA-42, and the fold of the template polypeptide is
preserved in spite of the replacement of five residues in the
sequence. A number of medium range NOEs typical of helical
conformations were observed in the segments 3 -18 and 28
Figure 5. Part of the NOESY spectrum of KO-42 in aqueous solution
at pH 5.8 and 323 K showing NOE contacts between Phe aromatic
protons and Ile, Leu, and Nle methyl protons. Connectivities between
Phe-35 and Phe-38 aromatic protons and the methyl group of Nle-5
demonstrate unequivocally the formation of the hairpin motif. The
chemical shifts deviate slightly from those in Table 1 since the solvent
is different.
-
41. The extent of helix formation is in agreement with the
20,21
magnitude of the mean residue ellipticity.
The short segment
between residues 18 and 26 is characterized by positive
deviations of the chemical shifts in comparison with those of
random coils which is typical of â structures, turns, and loops.
Because the lack of medium range NOEs suggests a floppy
structure these two observations indicate that this segment forms
a loop.
Long-range NOE contacts between the aromatic protons of
Phe35 and of Phe38 and the methyl groups of Nle-5 as well as
those of Ile-9 or Leu-12, or both, were identified (Figure 5)
and show the formation of the hairpin conformation as described
for SA-42.4 Close proximity between the upper end of helix I
and the lower end of helix II is only compatible with the
formation of a hairpin motif. It was established by equilibrium
sedimentation ultracentrifugation that KO-42 is a dimer at pH
11 000 D which is approximately 20% higher than the calculated
molecular weight of the dimer, 8980 D. The ultracentrifugation
experiment was also carried out at 303 K, but the difference
between the results at the two temperatures was small, less than
5
%. The combined results from NMR and CD spectroscopy
and ultracentrifugation therefore show that KO-42 folds into a
hairpin helix-loop-helix motif that dimerizes to form a four-
helix bundle. The interface between the two monomers within
the dimer contains mainly alanine residues which makes it hard
to identify intermolecular NOEs since alanine methyl groups
are not well dispersed and cross peaks appear close to the
diagonal in the NOESY spectrum. In the case of SA-42,
intermolecular NOEs were unequivocally assigned that showed
connectivity between Phe aromatic protons and methyl groups
6
and 293 K with evidence for some higher order aggregates.
The experimentally determined molecular weight of KO-42 was
(
22) Wishart, D. S.; Sykes, B. D.; Richards, F. M. J. Mol. Biol. 1991,
2
22, 311.

Hydrolysis/Transesterification Reactions of p-Nitrophenyl Esters
J. Am. Chem. Soc., Vol. 119, No. 47, 1997 11365
Table 1. Rate Constants for Acyl-Transfer Reactions of p-Nitrophenyl Esters at 290 K
k2(KO-42)
k2(KO-42)
k(uncat)
k
(
2
(KO-42)
2
k (4-MeIm)
-
1
-1
-1 -1
k2(4-MeIm)
M
s
)
(M
s
)
k(uncat)
Mono-p-nitrophenyl Fumarate
-
-
-
-
-
5
5
4
2
3
aq buf pH 4.1
0% TFE, pH 4.1
aq buf, pH 5.1
aq buf, pH 5.85
0.10
8.8 * 10
8.4 * 10
7.4 * 10
1.0 * 10
1.2 * 10
1140
620
420
65
1
0.052
0.31
0.65
0.064
0.68
0.10
1.2 * 10-6 M-1 s-1
43000
3
D
D
0% TFE, pH 5.85
3.7 * 10-6 M-1 s-1
54
2
O buf, pH* 6.1
O buf, pH* 4.7
2
p-Nitrophenyl Acetate
4
aq buf, pH 5.1
aq buf, pH 5.1
aq buf, pH 5.1
0.29
3.7
7.2 * 10-
402
670
D- and L-Tryptophane p-Nitrophenyl Ester
-
3
5.5 * 10
Cyclopentane-trans-1,2-dicarboxylic Acid Mono-p-Nitrophenyl Ester
-
3
0.86
1.5 * 10
590
Chart 1
in a hydrolysis reaction, and an acyl-transfer to the trifluoro-
2
3
ethoxide ion corresponds to a transesterification. Intramo-
lecular acyl transfer to a primary amine has been described
9
previously.
The reactions were conveniently followed by monitoring the
formation of p-nitrophenol by UV spectroscopy at 320 nm. They
followed excellent pseudo-first-order kinetics for more than five
half-lives even under conditions of more than a 10-fold excess
of p-nitrophenyl acetate, and no saturation kinetics were
observed in aqueous solution at pH 6.2. When the substrate
was mono-p-nitrophenyl fumarate some product inhibition due
to the interaction between fumarate mono- and dianions and
the partly protonated catalyst was detected in the kinetic
measurements. As a control experiment the reaction was run
at pH 5.85 with 10 vol % TFE in the presence of a 4-fold excess
of fumaric acid, mainly in its dianionic form, and the second-
order rate constant for the KO-42 catalyzed reaction was
decreased by about 20% in comparison with the rate constant
measured without added fumaric acid. A 4-fold excess of the
leaving group, p-nitrophenol, did not affect the reaction kinetics.
The observed rate decrease upon addition of fumaric acid agrees
well with the NMR spectroscopic observation that KO-42 binds
the fumaryl dianion.
The second-order rate constants for the reactions of mono-
p-nitrophenyl fumarate and p-nitrophenyl acetate were deter-
mined in all cases from the slopes of the linear plots of 3-6
pseudo-first-order rate constants versus concentration of catalyst.
In the presence of added NaCl or fumaric acid the reactions
were run only once, and their pseudo-first-order rate constants
were compared to those of the corresponding reactions without
added salt or acid. The pseudo-first-order rate constants for
the reactions of the tryptophane and cyclopentanedicarboxylic
acid esters were measured twice, and the rate constants for the
background reactions were substracted. Division of resulting
rate constants by the total concentration of catalyst gave the
second-order rate constant.
in the hydrophobic core and demonstrated antiparallel dimer-
ization. Since the sequence of KO-42 is the same as that of
SA-42 except for the replacement of 5 amino acid residues we
assume that the mode of dimerization is the same.
The Reactions Catalyzed by KO-42. KO-42 catalyzes acyl-
transfer reactions of p-nitrophenyl esters (Table 1) and the
products of the KO-42 catalyzed reactions of mono-p-nitrophen-
1
yl fumarate (I) were identified from the H NMR spectrum
recorded under reaction conditions. In aqueous solution the
fumaryl anions were identified as reaction products from the
1
single resonance in the H NMR spectrum, and the assignment
was verified by the addition of a few crystals of fumaric acid
to the NMR tube. At pH 5.5 the resonance was severely
broadened suggesting that the fumaryl dianion binds to the
polypeptide. Minor amounts, less than 5%, of Bis-Tris ester
were detected in 10 vol % TFE when Bis-Tris buffer was used,
but in acetate buffer the fumaryl anions were the only observable
products.
Under reaction conditions in 10 vol % TFE the spin system
of the substrate, the fumaryl monoester (two doublets, J ) 16
Hz), was replaced by that of the reaction product, a second
fumaryl spin system, which was found to be the corresponding
trifluoroethyl mono ester. KO-42 therefore catalyzes hydrolysis
reactions in aqueous solution and transesterification reactions
to form the trifluoroethyl ester in 10 vol % trifluoroethanol
(
TFE).
ES-MS was used to measure the molecular weight of the
peptide after reaction with a 10-fold excess of I at pH 6.1. About
0% of the peptide had become acylated by the fumaryl residue
2
The reaction was first order in catalyst which shows that the
dimer is the reactive species.
The second-order rate constants for the KO-42 catalyzed
hydrolysis and transesterification reactions of p-nitrophenyl
esters are presented in Table 1, together with those of the
probably through a direct reaction between the reactive ester
and a lysine side chain, and the same reaction is also likely to
occur with other p-nitrophenyl esters. The reactivity of KO-
4
2 was, however, not affected, and acylation does not perturb
the function of KO-42. CD spectroscopy was also used to
monitor the reaction under conditions of excess substrate, and
the mean residue ellipticity did not change. The reaction
products obtained were those expected in imidazole catalyzed
reactions where an initially formed acyl intermediate reacts with
the most potent nucleophile in the reaction solution to form the
reaction product. An acyl transfer to the hydroxide ion results
4
-methylimidazole catalyzed reactions. The rate constant ratio,
k2(KO-42)/k2(4-MeIm), is pH dependent, and its value at pH
.1 for the reaction with mono-p-nitrophenyl fumarate is 1140,
4
and at pH 5.1 it is slightly less than half of that, 420. The
difference is close to what is calculated from the Brønsted
(23) Oakenfull, D. G.; Jencks, W. P. J. Am. Chem. Soc. 1971, 93, 178.

11366 J. Am. Chem. Soc., Vol. 119, No. 47, 1997
Broo et al.
Chart 2
relation and the difference in pKa values of KO-42 and 4-MeIm
using a Brønsted â coefficient of 0.8, as for imidazole catalyzed
hydrolysis of p-nitrophenyl acetate.24 The rate enhancement
over background in 10 vol % TFE, i.e., over the “uncatalyzed”
noninteracting histidine side chains are expected to have pKa
values of 6.4, and the pH dependence was therefore surprising.
25
The pKas of the six histidines of KO-42 were determined
1
from the 1D H NMR spectrum at 319 K recorded as a function
5
hydrolysis reaction, is 0.43 × 10 , and the rate constant ratio,
of pH* in D2O solution because the resonances were too wide
k2(KO-42)/k2(4-MeIm), for the transesterification reaction is 620
in 10 vol % TFE at pH 4.1 and 290 K. These rate enhancements
are the largest reported so far for rationally designed four-helix
bundle catalysts and are comparable to those observed for typical
catalytic antibodies. A number of substrates have been used
for acyl-transfer reactions (Table 1), and the similarity in rate
enhancements suggests that the recognition of substrates by the
catalyst is poor, with regards to positively charged ammonium
ions, negatively charged fumaryl residues, and hydrophobic
cyclopentyl- and indolyl residues. Alternatively, too many
reaction pathways are available in KO-42 catalyzed reactions.
The reactivity of KO-42 toward p-nitrophenyl acetate can be
compared to its reactivity toward I to measure any interaction
between reactive site residues of KO-42 and the anionic fumaryl
residue of the substrate. The reactivity is very similar for both
substrates, and there is therefore little binding of the fumaryl
residue by the catalyst.
1
at 290 K to allow the assignment of the H NMR spectrum.
Imidazole proton chemical shifts of the histidine residues were
determined over a range of more than 6 pH units, and at pH
6
.1 they were compared to the assignments from the TOCSY
and NOESY spectra. A dissociation curve and a pKa could
therefore be assigned to each residue. The effect of temperature
on pKa was estimated to be negligible since the pKa determi-
nation of the 4-methylimidazolium ion at 319 and at 291 K
showed a difference that was less than 0.05 pKa units with the
lower pKa found at the lower temperature. The pKa values of
the histidine residues of KO-42 at 319 K were therefore used
in the analysis of the kinetic results at 290 K. The pH titration
experiment with KO-42 was also carried out at 291 K in 7 vol
%
TFE, and the titration curves gave pKa values that were within
1
0
.1 pKa units of the ones obtained at 323 K. But the H NMR
spectrum could not be assigned at 291 K due to line broadening,
and the pKa values could not be unequivocally assigned to
individual histidine residues at the lower temperature.
The pH Dependence of KO-42 Catalyzed Reactions and
the Determination of pKas for the Histidine Side Chains. The
dependence on pH of the reactivity of KO-42 was determined,
and the observed second-order rate constants showed a strong
depencence on pH (Figure 6). A function describing the
dissociation of a monoprotonic acid with a pKa of 5.1 gave the
best fit to the experimental results although this is obviously
an oversimplification, see Discussion. The side chains of
Clear deviations from ideal titration behavior were observed
(Figure 7), suggesting strong intramolecular interactions between
the histidines, in agreement with the observed pronounced
dependence of the mean residue ellipticity on pH. The pKa
values of His-15, His-30, and His-34 determined from the best
fits to the experimental results were 5.5, 5.4, and 5.3, respec-
tively, and those of His-11, His-19, and His-26 were 6.5, 6.6,
and 7.0 (Figure 7). The best fits of dissociation curves for
monobasic acids to the experimental results were obtained for
the more acidic histidines, whereas poorer fits were obtained
for the more basic histidine residues.
6
KO-42 can exist in 2 , or 64, different states of protonation
since each one of the six histidine residues can be either
protonated or unprotonated. The pH dependence of the CD
spectrum shows that different protonation states also have
different structures. The observation of nonideally titrating
histidines is in fact the observation of the titration of a large
number of species with different pKas. At low pH all histidines
are protonated, and as base is added the less basic ones start to
titrate, while the more basic ones are still mainly protonated.
His-15, His-30, and His-34 titrate, while His-11, His-19, and
His-26 are mainly protonated. At a pH where only a small
fraction of the more acidic histidines are unprotonated and the
other histidines are protonated, they titrate essentially indepen-
dently of each other. With increasing pH the less acidic ones
also titrate to a significant degree, and the latter part of the
titration curves of the more acidic ones reflect a composition
of a large number of protonation states that are present
simultaneously. With the application of a weighting function
that emphasizes the early part of the titration curve the obtained
pKa values were 5.4, 5.3, and 5.2 (Figure 8), and these pKa
values were used in the analysis of the pH dependence of the
rate constants.
Figure 6. Observed second-order rate constants versus pH for the KO-
4
2 catalyzed reaction of mono-p-nitrophenyl fumarate in aqueous
solution at 290 K. The solid line describes the dissociation of a
monoprotonic acid with a pK of 5.1. The function assumes that the
catalytic rate of completely protonated KO-42 is 0, but fitting a function
where the second-order rate constant is allowed to vary gives a pK
with a value that differs by only 0.04 pK units. The value of the second-
order rate constant for the hexaprotonated species obtained under those
a
a
a
-
1
-1
conditions was 0.02 M
s . The estimate of the second-order rate
constants at pH 4.7 and 6.1 for calculation of the kinetic solvent isotope
effects are indicated.

Hydrolysis/Transesterification Reactions of p-Nitrophenyl Esters
J. Am. Chem. Soc., Vol. 119, No. 47, 1997 11367
Figure 7. The experimentally determined dissociation curves of the six histidine residue side chains with the best fits to the experimental data of
a function describing the dissociation of a monoprotonic acid. Both the chemical shifts of the unprotonated and protonated histidines as well as the
dissociation constants were allowed to vary. The pK values were 5.3 for His-34 (A), 5.4 for His-30 (B), 5.5 for His-15 (C), 6.5 for His-11 (D), 6.6
a
for His-19 (E) and 7.0 for His-26 (F).
Strongly nonideal behavior is shown by the more basic
histidine residues, but they do not reach their titration midpoints
until a pH of almost 7 has been reached. Their pKas are
therefore a pKa unit or more higher than those of His-15, His-
adding 0.4 pH units to the reading on the pH meter, and the
D2O solution is therefore more alkaline than the H2O solution.
There is, however, also an equilibrium isotope effect on
dissociation constants which is mainly due to the fractionation
of the lyonium ion, and an acid is therefore less dissociated in
3
0, and His-34 for all the 64 species in solution. The best fits
to the experimental results were obtained for pKa values of 6.4,
.5, and 7.0 (Figure 7). Exact pKa values of His-11, His-19,
2
6
D2O. Since the isotopic fractionation of the imidazolium ion
27
6
can be neglected, these two effects approximately cancel, and
the reaction in H2O at a given pH can be compared to the
reaction in D2O with the same pH* since the fraction of
unprotonated catalyst is the same. The kinetic solvent isotope
and His-26 in all the 64 different species are obviously
impossible to measure, but the pKas of the monoprotonated
histidine residues in the limiting case when all other histidines
are unprotonated are of special interest in the discussion of the
nature of the reactive species in the pH independent region. A
fit of dissociation curves with weighting functions applied such
that the last part is emphasized gives pKa values of 6.9, 7.0,
and 7.2 for the three histidines (Figure 8). These values have
been used in the discussion of the identity of the species
responsible for the catalysis in the pH independent region.
2
6
effect is given by eq 1, where ∏φR is the product of
fractionation factors for all exchangeable hydrogens in the
†
k /k ) ∏φ /∏φ
(1)
H
D
R
†
reactant state and ∏φ is the corresponding product for the
transition state. Isotopic fractionation in exchangeable hydro-
gens arises when the potential well of the hydrogen deviates
substantially from that of the solvent, e.g., when it is strongly
hydrogen bonded.
The Kinetic Solvent Isotope Effect. The pH dependence
of the observed second-order rate constant for the KO-42
catalyzed hydrolysis of I showed that the reactive species at
low pH had acidic and partly unprotonated histidine residues
flanked by more basic and mainly protonated histidines. Further
investigations of the role of the flanking residues were therefore
carried out by measurements of the kinetic solvent isotope effect
for the KO-42 catalyzed hydrolysis of I at pH 4.7 and at pH
The kinetic solvent isotope effect of the KO-42 catalyzed
reaction at pH 4.7 is 2.0, and isotopic fractionation must
therefore occur in the transition state. Strong hydrogen bonding
and/or general acid-general base catalysis is therefore suggested
to contribute to the observed rate enhancement. At pH 6.1 the
kinetic solvent isotope effect is 1.1 which shows that there is a
6.1 and 290 K. The second-order rate constants in D2O solution
(
Table 1) were measured at the reported pH* values, whereas
(
24) Bruice, T. C.; Lapinski, R. J. Am. Chem. Soc. 1958, 80, 2265.
(25) Tanford, C. AdV. Protein Chem. 1962, 17, 69.
26) Laughton, P. M.; Robertson, R. E. Solute-SolVent Interactions;
Coetzee, J. F., Ritchie, C. D., Eds.; Marcel Dekker: New York, 1969; pp
the corresponding second-order rate constants in H2O solution
were estimated as shown in the plot of k2 versus pH (Figure 6).
The pH* in D2O solution was determined by a glass electrode,
and according to the usual convention the pD is obtained by
(
3
99-525.
(27) Li, N. C.; Tang, P.; Mathur, R. J. Phys. Chem. 1961, 65, 1074.

11368 J. Am. Chem. Soc., Vol. 119, No. 47, 1997
Broo et al.
Figure 8. The experimentally determined dissociation curves of the six histidine residue side chains with the best fits to the experimental data of
a function describing the dissociation of a monoprotonic acid with weighted points at the early and late stages of dissociation. The pK values were
.2 for His-34 (A), 5.3 for His-30 (B), 5.4 for His-15 (C), 6.9 for His-11 (D), 7.0 for His-19 (E), and.7.2 for His-26 (F).
a
5
change of reaction mechanism with pH. No contributions from
general base-general acid catalysis or strong hydrogen bonding
in the transition state can be observed for the reaction at pH
Discussion
KO-42 catalyzes acyl-transfer reactions of p-nitrophenyl esters
with second-order rate constants that are more than 10 times
3
6
.1. The measured kinetic solvent isotope effect at pH 6.1 is
larger than those of 4-MeIm in aqueous solution. The rate
enhancements are comparable to those of typical catalytic
antibodies, and the structure and function of the catalyst is
therefore of great interest for the further development of de noVo
designed polypeptide catalysts.
the same as that reported for the imidazole catalyzed hydrolysis
of p-nitrophenyl acetate.28
Salt and Solvent Effects on the Reactivity of KO-42. The
second-order rate constant determined in 1.0 M NaCl at pH
5
.8 was the same, within experimental error, as that determined
The Structure of the Catalyst. KO-42 folds into a hairpin
helix-loop-helix dimer as demonstrated by NMR and CD
spectroscopy and equilibrium sedimentation ultracentrifugation.
Two helical segments and a loop were identified from the R
proton chemical shifts and the observed medium-range NOEs
(Figure 4), and the magnitude of the mean residue ellipticity at
222 nm was used to calculate a helical content that was
compatible with the extension of the helical sequences. Long-
range NOEs were used to identify the formation of the hairpin
motif (Figure 5), and dimerization was demonstrated by
ultracentrifugation. The formation of the hairpin helix-loop-
helix motif has been found in all the peptides developed from
SA-42 and is not surprising since the residues that form the
hydrophobic core of the folded four-helix bundle have been
preserved.
in aqueous solution without added salt. The second-order rate
constants for the KO-42 and 4-MeIm catalyzed reactions were
also measured at pH 5.8 in 30 vol % TFE, a less polar solvent
than water, and the second-order rate constants were decreased.
The ratio of the second-order rate constants, k2(KO-42)/k2(4-
MeIm), was, however, roughly the same, and charge-charge
interactions in the transition state were apparently unimportant
or equally important as those in the ground state. In contrast,
the relative reactivity of the helix-loop-helix motif RA-42
toward I, k2(RA-42)/k2(4-MeIm), was enhanced by almost an
order of magnitude in 30 vol % TFE.17 Flanking histidine
residues therefore have a different function at pH 6.1 than
flanking Arg, Lys, and Orn.
The effect of the solvent on the structure of SA-42 has been
determined previously,16 and in 30 vol % TFE SA-42 is
The magnitude of the mean residue ellipticity of KO-42 is
4
together with that of SA-42 the largest reported so far for a
predominantly a nonhairpin, helix-loop-helix monomer. Since
the magnitude of the rate constant ratio does not decrease more
than approximately 20%, one can conclude that the reactive sites
are contained within a single helix. It is not yet possible to
conclude whether there is one or more reactive sites in KO-42.
helix-loop-helix motif of this size, and the addition of 16 vol
%
of the helix inducing solvent TFE does not increase the helical
content by more than 10%. The amount of helix formation in
KO-42 does not allow for the existence of disordered segments
in the two helices so the secondary structures are well defined.
The supersecondary structure formation is usually monitored
by measuring the concentration dependence of the mean residue
(28) Bender, M. L.; Pollock, E. J.; Neveu, M. C. J. Am. Chem. Soc.
1
962, 84, 595.

Hydrolysis/Transesterification Reactions of p-Nitrophenyl Esters
J. Am. Chem. Soc., Vol. 119, No. 47, 1997 11369
1
ellipticity, because if the helix-loop-helix dimers dissociate
to form monomers its magnitude will decrease. The observation
of large negative values for concentrations of KO-42 above 200
µM therefore means that the fraction of monomers is small and
that the supersecondary structure of KO-42 is well-defined
although the hydrophobic core is not as well packed as that of
a native protein. The solution structure of SA-42, the template
polypeptide, has been discussed in a similar vein in detail,
reaction approaches a constant value of 1.3 M- s-1, and the
rate constant ratio at high pH is less than 1. The difference in
reactivity of the two catalysts is related to the difference in pKa,
as described by the Bronsted relation (eq 2). The pKa of
4-MeIm is 7.9 and the pKas of the six histidines of KO-42 in
(2)
2
log k ) A + âpKa
4
the pH independent region are 5.2, 5.3, 5.4, 6.9, 7.0, and 7.2.
The second-order rate constant for a reaction catalyzed by a
peptide with six histidine residues may be estimated from the
second-order rate constant of 4-MeIm using the pKa values of
-MeIm and the six histidines, the Brønsted relation, and a
coefficient â of 0.8, which is the value for imidazole-catalyzed
previously.
At low pH, the helical content of KO-42, unlike that of SA-
42, is severely reduced (Figure 3), and the introduced histidine
residues, when protonated and positively charged, probably repel
each other strongly. The mean residue ellipticity at pH 2.5 is
4
2
-1
only -5000 deg cm dmol which is a value typically found
for disordered monomers or random coils with some helix
propensity. At high pH when the histidine side chains are
largely unprotonated and do not carry any charges, the helical
content is high.
2
4
p-nitrophenyl acetate hydrolysis. Six second-order rate con-
stants were calculated for histidines with the pKa values of KO-
-
1 -1
4
2, and the sum of the rate constants was 0.7 M
s
which is
-
1
very close to the experimentally determined value of 0.74 M
-
1
s
for the KO-42 catalyzed reaction. In the pH independent
The observation of perturbed pKa values in KO-42 suggests
that the histidines interact. When all histidines are unprotonated,
the helical content is the highest probably because there is no
repulsion due to charge-charge interactions and no intramo-
region the reactive site of KO-42 therefore functions as six
independent histidine residues, and there is no rate acceleration
due to cooperative effects between more than one residue.
At pH 4.1 the rate constant ratio in aqueous solution at 290
K is 1140. The magnitude of the observed rate constant ratio
is partly due to the difference in pKa between 4-MeIm (7.9)
and the His side chains of KO-42 (5.2 -5.4). The concentration
of free base at low pH for 4-MeIm is smaller than for KO-42,
but the intrinsic reactivity of 4-MeIm is higher due to its higher
pKa. The net effect at a given pH can be calculated from the
Brønsted relation using the coefficient â and the pKa values of
the catalysts. For a â that is less than 1, and a pH that is less
than the pKa values of both catalysts, a weaker base will be a
more efficient nucleophile than a stronger base if the total
concentrations of catalysts (N + NH ) are the same. From
the pKa values of KO-42 and of 4-MeIm and from the Brønsted
relation it can be calculated that for the hydrolysis of p-
nitrophenyl fumarate at pH 4.1 the KO-42 catalyzed reaction
is favored only by a factor of 3.2 over that of 4-MeIm due to
the lower degree of protonation. A catalytic factor of 360 is
therefore due to transition state binding, assuming that a single
dominant catalytic site is active that contains only one of the
+
lecular HisH -His hydrogen bonding. The secondary structure
of KO-42 is the most stabilized when they are all unprotonated.
+
+
As a result of HisH -HisH interactions the mean residue
ellipticity at pH 4.1 is only approximately 65% of that at pH 7.
The structure of the reactive species is probably not affected
since it contains unprotonated histidines, flanked by protonated
histidines, and little repulsion from charge-charge interactions.
This is reflected in the magnitude of the second-order rate
constant which follows the function describing the dissociation
of an acid quite well. It is, however, also possible that it is the
neutralizations of the charged aspartates and glutamates that lead
to lower helical content.
+
29
In the designed motif the histidine residues are located in
helical conformations, and each helix contains three residues
organized in an almost straight line with a distance between
the R carbons of 5.4 Å. It can, however, be seen from the R
proton chemical shifts that in the solution structure His-19 is
located in the loop region (Figure 4). Its position relative to
that of His-15 may therefore not be well defined. His-26 is
found in the first turn of helix II, and its orientation relative to
His-30 is probably as designed. In helix I a His residue with a
depressed pKa is flanked by two histidines with increased pKas.
In helix II two neighboring residues with depressed pKa values
are flanked on one side by a histidine side chain with an
increased pKa. The organization of unprotonated and protonated
His residues as a function of pH is crucial to the reactivity of
KO-42.
low-pK histidines.
a
The reactivity of KO-42 toward the p-nitrophenyl esters of
acetic acid (II), tryptophane (III), and cyclopentane-trans-1,2-
dicarboxylic acid (IV) was also investigated in aqueous solution
at pH 5.1 (Table 1) to explore the possibility of substrate
recognition by KO-42. The second-order rate constants vary
within a factor of 10, but the rate constant ratio, k (KO-42)/
k2(4-MeIm), stays relatively constant in the range from 402 to
2
670 (Table 1). Binding of the anionic fumaryl residue by the
cationic KO-42 would seem likely, but the results do not support
such a conclusion as the rate constant ratio is virtually the same
for the fumaryl and the acetyl residues. In the tryptophane ester
there is a positively charged ammonium group in close proximity
to the ester function, but catalysis of the tryptophane ester
hydrolysis is not less efficient than that of the fumaryl ester
since the rate constant ratio is 670. The catalytic efficiency
seems to be rather insensitive to the presence of charged residues
in the acetyl residue of the substrate, and it is likely that the
charged groups are oriented away from the positively charged
flanking residues into the solvent. The reactive site of KO-42
is perhaps instead capable of recognizing hydrophobic groups,
since the largest ratios are those of the tryptophane esters and
of the cyclopentanedicarboxylic acid monoester. These results
The Reactivity of KO-42. KO-42 catalyzes acyl-transfer
reactions of p-nitrophenyl esters in aqueous solution and in
mixtures of water and TFE to form the corresponding carboxylic
acids and TFE esters. Because the reactive site of KO-42
contains His residues the second-order rate constants of the KO-
4
4
2 catalyzed reactions have been compared to those of the
-MeIm catalyzed reactions (Table 1), and in aqueous solution
the rate enhancement at pH 4.1 is larger than three orders of
magnitude. The second-order rate constants and the second-
order rate constant ratio, k2(KO-42)/k2(4-MeIm), vary with pH,
and in order to assess the efficiency of the catalyst the
dependence of rate on pH has been investigated.
The observed second-order rate constant of the KO-42
catalyzed reaction of I increases with pH (Figure 6) and
-
1
-1
approaches asymptotically a constant value of 0.74 M s .
The second-order rate constant for the 4-MeIm catalyzed
(
29) Fersht, A. Enzyme Structure and Mechanism; W. H. Freeman and
Co.: New York, 1985; pp 47-97.

11370 J. Am. Chem. Soc., Vol. 119, No. 47, 1997
Broo et al.
suggest that catalysts capable of chiral recognition of substrates
with hydrophobic groups may be engineered in de noVo designed
proteins.
reaction of I in aqueous solution, since the rate constant ratio
is 1, and general-base catalysis is therefore excluded at that pH,
in spite of the fact that there are many unprotonated histidine
residues available. The absence of an isotope effect at pH 6.1
shows that there is no fractionation in the transition state and
not in the ground state unless they happen to cancel exactly,
which excludes the possibility of strong hydrogen bonding
between protonated histidine side chains and the developing
oxyanion. It shows that at pH 6.1 the dominant reaction
mechanism is that of imidazole-like noncooperative catalysis,
which is what is found for the hexaunprotonated KO-42.
At pH 4.7 the kinetic solvent isotope effect is 2.0, and there
is substantial fractionation in the transition state of the reaction.
The observed effect shows that the reaction mechanism is
different from that at pH 6.1 and that hydrogens are being
transferred or hydrogen bonds are being formed or broken in
the transition state since a kinetic solvent isotope effect kH/kD
that is larger than 1 requires that the fractionation in the
transition state is more pronounced than that in the ground state.
Fractionation in the transition state is commonly used as
evidence for general base catalysis where the water molecule
that provides the hydroxide ion is hydrogen bonded to the
general base. Since no general base catalysis occurs at pH 6.1,
then there is little reason to assume that it does at pH 4.7 where
the fraction of unprotonated histidine residues capable of general
base catalysis is much smaller. General acid catalysis must
however be taken into account as a plausible catalytic mecha-
nism at pH 4.7, perhaps in terms of leaving group protonation.
The formation of a strong hydrogen bond to the developing
oxyanion is also compatible with the observed isotope effect.
The Function of KO-42. KO-42 catalyzes acyl-transfer
reactions of p-nitrophenyl esters, and the rate enhancements over
the 4-MeIm catalyzed reactions and the reaction mechanism of
KO-42 change with pH. At low pH the histidine side chains
of KO-42 are protonated to large extents, and the catalytically
active species have unprotonated, nucleophilic, histidine side
chains flanked by protonated side chains. The pH dependence
of the second-order rate constant shows that unprotonated
histidines are necessary for the catalytic activity, and the kinetic
solvent isotope effect shows that exchangeable hydrogens are
strongly hydrogen bonded in the transition state. The initial
rate-limiting step of the KO-42 catalyzed reaction at low pH is
therefore a nucleophilic attack by a unprotonated His side chain
on the carbonyl carbon of the ester to form an acyl intermediate.
Transition state binding of one or both of the ester oxygens is
provided by a flanking protonated histidine residue. In a second
step the acyl intermediate reacts with the most potent nucleophile
present to form the reaction product. Under these conditions
the rate enhancements are three orders of magnitude over that
of the 4-MeIm catalyzed reaction. No acyl intermediate has
been detected, in agreement with the fact that spontaneous
The pH Dependence of the KO-42 Catalyzed Reaction.
6
There are six histidine residues in KO-42 and therefore 2 or
6
4 different species resulting from all possible combinations
of protonated and nonprotonated histidine residues. The plots
of chemical shifts of the histidine side chain protons versus pH
show that the more acidic ones titrate with pKa values of 5.5,
5
.4, and 5.3 if the best fit of the function describing the
dissociation of an acid to the experimental data is used (Figure
). If weighting functions are applied that emphasize the early
7
parts of the dissociation curve at low pH then these values are
decreased by only 0.1 pKa units and values of 5.4, 5.3, and 5.2
are obtained instead (Figure 8). At pH 4.1 these residues titrate
essentially independently of each other since the probability of
finding two unprotonated histidine residues in the same polypep-
tide is less than 10% of the probability of finding a monoun-
protonated catalyst. The dominant polypeptides at pH 4.1 are
therefore, besides the nonreactive hexaprotonated one, three that
are pentaprotonated, and they exist in roughly equal amounts
due to their similar dissociation constants. The correlation of
the reaction rates with the concentration of the pentaprotonated
forms of KO-42 show that these are the catalytically active
species at low pH. In principle it is possible that all three
polypeptides are equally reactive, in which case the reactivity
per site is one third of that of KO-42.
Little can be said about the pKas and reactivities of most of
the close to 60 different species that coexist in the pH interval
from 5 to 7, except that their combined reactivities appear to
add up to a fairly constant value. It is probable that they
function through a combination of noncooperative imidazole-
like catalysis and cooperative catalysis by flanking protonated
and unprotonated histidine residues. The pH dependence for
the latter reaction mechanism should give rise to a bell-shaped
pH profile because it depends on the coexistence of a nucleo-
philic histidine in its unprotonated form and a binding histidine
in its protonated form. No bell-shaped pH profile was observed,
and the explanation is due to the reactivity of the hexaunpro-
tonated form of KO-42.
In the pH independent region in aqueous solution the histidine
residues of KO-42 are completely unprotonated, and the pKa
values of the more basic residues were estimated from the latter
part of the strongly perturbed dissociation curves through the
application of an appropriate weighting function. At high pH
the hexaunprotonated species must be the reactive species since
it is the only one that is avaliable in significant amounts. Its
reactivity can therefore be reliably determined from direct kinetic
measurements, and the concentration of that species is readily
calculated from the pKa values of the more basic histidine
residues. At a pH above 7.2 it approaches asymptotically the
total concentration of peptide, but for pH values below 6.9 it
drops off with a rate that approaches the cube of the hydrogen
ion concentration. At pH 5.1 the concentration of hexaunpro-
tonated catalyst is in the nM range and catalysis by the
hexaunprotonated catalyst is therefore negligible in comparison
with that by partially protonated peptides. If the fraction of
reaction catalyzed by the hexaunprotonated polypeptide is
subtracted from the plot of the second-order rate constant versus
pH the resulting pH profile shows a shape that approximates a
bell.
3
0
hydrolysis of acetylimidazole takes place faster than KO-42
catalyzed hydrolysis of I at the concentrations of catalyst used
here. Since the first step is rate limiting, the intermediate does
not accumulate.
At high pH the reactivity of KO-42 is independent of pH,
the second-order rate constant is very close to what is expected
from six independent histidine residues and there is no kinetic
solvent isotope effect as kH/kD is 1 within experimental error.
The reaction mechanism is therefore the same as that of
imidazole catalysis of ester hydrolysis, and no cooperativity
The Kinetic Solvent Isotope Effect. The kinetic solvent
isotope effect is a measure of whether isotopic fractionation of
exchangeable hydrogens occur in the reactant and/or transition
states. There is no effect at pH 6.1 in the KO-42 catalyzed
(30) Oakenfull, D. G.; Salvesen, K.; Jencks, W. P. J. Am. Chem. Soc.
1
971, 93, 188-194.
(
31) McMeekin, T. L.; Marshall, K., Science 1952, 116, 142.
(32) Wutrich, K. NMR of Proteins and Nucleic Acids; Wiley: New York,
1985.

Hydrolysis/Transesterification Reactions of p-Nitrophenyl Esters
J. Am. Chem. Soc., Vol. 119, No. 47, 1997 11371
between residues is found since there is no rate enhancement
over the imidazole catalyzed reaction.
The pK
a
values were measured at 319 K (calibrated) by determining
the chemical shifts of the H-2 and H-4 protons of the histidine side
chains as a function of pH* (uncorr.) in D O solution under the usual
assumption that the isotope effects cancel out. The experimentally
determined dissociation curves formed well resolved plots, and the
chemical shifts obtained at pH 6.1 were used to assign the titration
curves to individual histidine residues by comparison with the chemical
2
It is not possible to demonstrate whether all three histidine
side chains with depressed pKa values are catalytically active
since their pKa values are very close. If they are all active, the
rate enhancement in acyl-transfer reactions due to a nucleophilic
+
His flanked by a HisH is a factor of 120 over that of a single
1
shift assignment at that pH. The H NMR spectrum was assigned in
His, and a further factor of 3.2 stands to be gained if pKa values
can be designed such that they are depressed as in KO-42. The
combination of two histidine residues therefore shows great
promise as a concept around which more sophisticated reactive
sites can be engineered in the de noVo design of novel catalysts.
Optimism is now called for in the engineering of catalysts
capable of substrate recognition, chiral discrimination, and
catalysis of reactions for which there are no natural enzymes.
The observed rate enhancements are large enough to make
screening or selection methods a viable alternative for the further
development toward larger rate enhancements and specificity.
6
vol % TFE and partially in 2 and 4 vol %, and the small observed
chemical shift changes upon addition of 2, 4, and 6 vol % (1.8 mol%)
demonstrated that the assignment in 6 vol % TFE could be extrapolated
to aqueous solution. The temperature dependence of the pK
a
of
+
4
-MeImH was tested by measuring the dissociation constant at 319
a
and 291 K, and the obtained values differed by less than 0.05 pK units.
Samples for NMR spectroscopy were prepared by dissolving lyophilized
peptide in the appropriate solvent and adjusting the pH by addition of
0
.1 M NaOD or 0.1 M DCl as appropriate. TFE was added by
autopipette, and the solution was centrifuged to remove unsoluble
impurities. In the TFE titration study the pH was measured in aqueous
solution before addition of TFE. The pH is increased by addition of
the cosolvent by a few tenths of a pH unit, but the pH was not corrected
since the effect of pH on the spectra was negligible.
Conclusion
The pK
a
values were determined by fitting an equation describing
We have demonstrated the rational design of a four-helix
bundle catalyst capable of catalyzing acyl-transfer reactions of
p-nitrophenyl esters with rate enhancements that are comparable
to those of typical catalytic antibodies. The solution structure
has been determined by NMR and CD spectroscopy and
ultracentrifugation. The enhanced reactivity is due to cooper-
ativity of unprotonated and protonated histidine residues in a
helical segment of the helix-loop-helix dimer. The catalytic
efficiency is due to a simple combination of His residues which
may prove to be an important concept in the further development
of novel catalysts capable of substrate recognition and chiral
discrimination.
the dissociation of a monoprotonic acid to the plots of the chemical
shifts of the aromatic protons of the His residues versus pH*. The
dissociation constant was varied until the best fit to the experimental
results was obtained using the Igor Pro software (Wavemetrics Inc.).
In order to emphasize the early part of the dissociation curve, the first
three and the last two data points were given a weighting coefficient
of 3, whereas the other two were not weighted, i.e., a weighting
coefficient of 1 was applied. In the fits where the last part of the
dissociation curve was emphasized the first two and the last three data
points were given the weighting coefficient 3, whereas the other data
points were unweighted. The extreme points were used since they
provided the chemical shifts of the fully protonated and fully unpro-
tonated species.
CD spectra were recorded on a Jasco-720 spectrometer in the
wavelength interval 280 to 200 nm, and the instrument was routinely
calibrated with (+)-camphor-10-sulfonic acid.19 Samples were prepared
by diluting aqueous stock solutions by pipetting, and the concentrations
of peptide were determined by quantitative amino acid analysis of
samples from the stock solutions. The experiments were carried out
in 0.1, 0.5, and 1 mm cells, and the temperature dependence of the
mean residue ellipticity was measured using a water-jacketed cell
together with a circulating HETO constant temperature bath. Sodium
acetate buffer was used below pH 5.85, and Bis-Tris buffer was used
at pH 5.85 and above, except in the pH titration and temperature
experiments where no buffer was used.
Kinetic runs were carried out using Varian Cary 1 and Cary 4
spectrophotometers equipped with Varian temperature controllers by
following the absorbance at 320 nm. The kinetic runs with excess
substrate were followed at 405 nm due to the strong absorbance at 320
nm. The samples were prepared by dissolving the lyophilized peptide
in the reaction solvent, adjusting the pH, and centrifuging the solution
prior to transferring it to a 0.1 mm cuvette and typically 300 µL of
0.2-0.4 mM peptide solutions were used.
For kinetic runs with the fumarate and cyclopentanedicarboxylate
esters the substrates were weighed and dissolved in the reaction solvent
and 20 µL of substrate stock solutions were transferred to the peptide
solution. The measurements were started after brief shaking of the
cuvette and reintroduction in the thermostatted cell compartment. The
p-nitrophenyl acetate was poorly soluble in water, and it was therefore
dissolved in acetonitrile after which 2 µL of the stock solution was
transferred by autopipette to 315 µL of peptide solution. The D- and
L-tryptophane esters were dissolved in acetonitrile/buffer 50/50 after
which 2 µL was transferred to 315 µL of peptide solution in the cuvette.
Substrate concentrations were typically 0.1 mM except under conditions
of excess substrate over peptide. The peptide was prepared in a stock
solution, and the concentration of that was determined by quantitative
amino acid analysis.
Experimental Section
The synthesis was first attempted with t-BOC chemistry but with
poor results and as a consequence it was instead carried out using
FMOC protection groups. The peptide was synthesized on an
automated peptide synthesizer from Applied Biosystems using the
standard fast FMOC protocol. The amino terminal was capped by acetic
anhydride and the carboxy terminal was amidated. The peptide was
cleaved from the resin using a mixture of trifluoroacetic acid (4.5 mL),
thioanisole (250 µL), ethaneditiol (150 µL), and anisole (100 µL) at
room temperature for 2 h. It was precipitated by cold diethyl ether
and collected by centrifugation. The peptide was purified by isochratic
reversed-phase HPLC on a C-8 semipreparative Kromasil, 7 µ column
using 40% isopropyl alcohol and 60% 0.1% TFA, with a flow rate of
5
mL/min. KO-42 eluted as a single peak, and its purity was checked
by analytical HPLC on a 5µ Kromasil column under identical conditions
except for the flow rate which was 0.5 mL/min. The polypeptide was
identified by electrospray MS (calculated 4490, found 4490.6), and
the mass spectrum obtained provided a further check on the purity of
the peptide since no other peptide peaks were observed.
1
1
D, NOESY, and TOCSY H NMR spectra were recorded at 500
MHz using a Varian Unity 500 NMR spectrometer equipped with a
matrix shim system MHU 303 from Resonance Research Inc. NOESY
and TOCSY spectra were recorded in 90% H
2 2
O/10% D O and varying
amounts of TFE-d
in each increment. The NOESY mixing time was 200 ms, and the
TOCSY spin lock was 60 ms. The data were processed using linear
3
at 323 K with 2 × 256 increments and 16 transients
1
prediction and zero filled to 4096 × 2048 data points. The H NMR
spectrum was assigned in 6 vol % TFE and partially assigned in 2 and
4
vol % TFE to resolve ambiguities.
The apparent molecular weight was determined from the sedimenta-
tion equilibrium in a MSE Centriscan analytical ultracentrifuge using
a photoelectric scanner at 254 nm. The experimental molecular weight
was corrected for calibrated nonlinearity in the detection system and
electrostatic repulsion in the salt-free solution. The partial specific
The kinetic runs were followed for more than two half-lives and
the pseudo-first-order rate constants were determined by fitting a single
3
1
volume was calculated from the amino acid composition.

11372 J. Am. Chem. Soc., Vol. 119, No. 47, 1997
Broo et al.
exponential function to the experimental data using Igor Pro software.
The second-order rate constants for the reactions of mono-p-nitrophenyl
fumarate and p-nitrophenyl acetate were determined by linear regression
analysis of the experimentally measured pseudo-first-order rate constants
as a function of peptide concentration. The reactions of the tryptophane
and cyclopentanedicarboxylate esters were determined by substracting
the background reaction from the measured pseudo-first-order rate
analysis, and error limits for individual rate constants are probably
(10%.
Acknowledgment. We are indebted to Dr. Christin Choma
for the FMOC synthesis of a batch of the peptide and to Dr.
Gunnar Johansson for the ultracentrifugation measurements.
Financial support from Carl Tryggers Stiftelse and the Swedish
Natural Science Research Council is gratefully acknowledged.
+
constant and dividing by the total concentration of peptide (N + NH ).
The concentration of peptide was determined by quantitative amino
acid analysis. The errors in the rate constants are due to the errors in
the regression analysis and the accuracy of the quantitative amino acid
JA970854S