Using unnatural amino acids to probe the energetics of oxyanion hole hydrogen bonds in the ketosteroid isomerase active site

Article abstract of DOI:10.1021/ja413174b

Hydrogen bonds are ubiquitous in enzyme active sites, providing binding interactions and stabilizing charge rearrangements on substrate groups over the course of a reaction. But understanding the origin and magnitude of their catalytic contributions relative to hydrogen bonds made in aqueous solution remains difficult, in part because of complexities encountered in energetic interpretation of traditional site-directed mutagenesis experiments. It has been proposed for ketosteroid isomerase and other enzymes that active site hydrogen bonding groups provide energetic stabilization via "short, strong" or "low-barrier" hydrogen bonds that are formed due to matching of their pK_a or proton affinity to that of the transition state. It has also been proposed that the ketosteroid isomerase and other enzyme active sites provide electrostatic environments that result in larger energetic responses (i.e., greater "sensitivity") to ground-state to transition-state charge rearrangement, relative to aqueous solution, thereby providing catalysis relative to the corresponding reaction in water. To test these models, we substituted tyrosine with fluorotyrosines (F-Tyr's) in the ketosteroid isomerase (KSI) oxyanion hole to systematically vary the proton affinity of an active site hydrogen bond donor while minimizing steric or structural effects. We found that a 40-fold increase in intrinsic F-Tyr acidity caused no significant change in activity for reactions with three different substrates. F-Tyr substitution did not change the solvent or primary kinetic isotope effect for proton abstraction, consistent with no change in mechanism arising from these substitutions. The observed shallow dependence of activity on the pK_a of the substituted Tyr residues suggests that the KSI oxyanion hole does not provide catalysis by forming an energetically exceptional pK_a-matched hydrogen bond. In addition, the shallow dependence provides no indication of an active site electrostatic environment that greatly enhances the energetic response to charge accumulation, consistent with prior experimental results.

Full text of DOI:10.1021/ja413174b

Article
pubs.acs.org/JACS
Using Unnatural Amino Acids to Probe the Energetics of Oxyanion
Hole Hydrogen Bonds in the Ketosteroid Isomerase Active Site
Aditya Natarajan, Jason P. Schwans,† and Daniel Herschlag*
Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305, United States
S
* Supporting Information
ABSTRACT: Hydrogen bonds are ubiquitous in enzyme
active sites, providing binding interactions and stabilizing
charge rearrangements on substrate groups over the course of
a reaction. But understanding the origin and magnitude of
their catalytic contributions relative to hydrogen bonds made
in aqueous solution remains difficult, in part because of
complexities encountered in energetic interpretation of
traditional site-directed mutagenesis experiments. It has been
proposed for ketosteroid isomerase and other enzymes that active site hydrogen bonding groups provide energetic stabilization
via “short, strong” or “low-barrier” hydrogen bonds that are formed due to matching of their pKa or proton affinity to that of the
transition state. It has also been proposed that the ketosteroid isomerase and other enzyme active sites provide electrostatic
environments that result in larger energetic responses (i.e., greater “sensitivity”) to ground-state to transition-state charge
rearrangement, relative to aqueous solution, thereby providing catalysis relative to the corresponding reaction in water. To test
these models, we substituted tyrosine with fluorotyrosines (F-Tyr’s) in the ketosteroid isomerase (KSI) oxyanion hole to
systematically vary the proton affinity of an active site hydrogen bond donor while minimizing steric or structural effects. We
found that a 40-fold increase in intrinsic F-Tyr acidity caused no significant change in activity for reactions with three different
substrates. F-Tyr substitution did not change the solvent or primary kinetic isotope effect for proton abstraction, consistent with
no change in mechanism arising from these substitutions. The observed shallow dependence of activity on the pKa of the
substituted Tyr residues suggests that the KSI oxyanion hole does not provide catalysis by forming an energetically exceptional
pKa-matched hydrogen bond. In addition, the shallow dependence provides no indication of an active site electrostatic
environment that greatly enhances the energetic response to charge accumulation, consistent with prior experimental results.
differs from bulk solution and may also be altered relative to the
environment of the wild type enzyme.3 Comparisons between
wild type and mutant enzymes are therefore complex, and the
rate effects from mutations cannot simply be interpreted as an
energetic contribution to wild type catalysis from the removed
group.3,4
Another level of complexity in understanding the contribu-
tion of hydrogen bonds to enzymatic catalysis arises because
their free energies of formation are highly dependent on the
surrounding environment.5−7 For example, while the free
energy change upon hydrogen bond formation between
fluoride ion and hydrogen fluoride is −0.8 kcal/mol in water,
in the gas phase the formation energy is estimated to be
between −32 to −39 kcal/mol.8−10 The surrounding solvent
can also affect the sensitivity of hydrogen bond energetics to
changes in the electrostatic properties of the hydrogen bond
donor and acceptor. For example, the dependence of the free
energy of hydrogen bond formation on the difference in pKa
(ΔpKa) between the donor and acceptor for a series of
substituted salicylates is shallow in water but is 15-fold steeper
INTRODUCTION
■
Hydrogen bonds are a common feature of enzyme active sites,
and their energetic contribution to catalysis has been the
subject of much discussion. Most basically, when hydrogen
bonds are made to functional groups that undergo charge
rearrangement over the course of a reaction, their strength is
expected to change in going from the ground state to the
transition state. The classic example in enzyme catalysis is that
of the serine protease oxyanion hole, in which hydrogen bonds
are donated to the oxygen atom of a carbonyl group that
accumulates charge as the reaction proceeds to the transition
state and the oxyanion intermediate.1,2 These hydrogen bonds
can contribute energetically to catalysis if their net strengthen-
ing in going from the ground state to the transition state
exceeds that for the hydrogen bonds formed to water in the
corresponding nonenzymatic reaction in aqueous solution.
The importance of active site hydrogen bonds is highlighted
by the plethora of site-directed mutagenesis experiments that
have demonstrated deleterious effects upon the removal of
active site hydrogen bond donors and acceptors.3 However,
traditional mutagenesis replaces hydrogen-bonding groups with
hydrophobic side chains and/or “holes” and can further be
accompanied by structural rearrangements and the introduction
of water molecules within an idiosyncratic environment that
Received: December 27, 2013
Published: May 1, 2014
© 2014 American Chemical Society
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Figure 1. Mechanism of KSI-catalyzed isomerization. KSI uses a general base Asp40 to isomerize double bonds adjacent to carbonyl groups through
a dienolate intermediate that is stabilized by the oxyanion hole formed from the Tyr16 and Asp103 side chains. Residues are numbered according to
their sequence position in KSI from Pseudomonas putida, the enzyme used in this study.
Figure 2. Semisynthesis of KSI with substituted tyrosines in the oxyanion hole. (A) Structures, abbreviations, and side chain pKa values of inserted
tyrosines. The pKa values are for the corresponding phenols.86,87 (B) Substituted tyrosines were protected and incorporated into a 23-mer synthetic
peptide with a C-terminal thioester. The remainder of the protein was expressed as a SUMO fusion, followed by removal of the N-terminal SUMO
tag and ligation to the synthetic peptide to generate full-length protein. (C) Electrospray mass spectra of semisynthetic (black) and recombinant
(gray) WT enzyme (i), and semisynthetic enzymes with substituted tyrosines (ii−v). The expected masses for KSI with Tyr, 2-F- or 3-F-Tyr, 2,6-F2-
Tyr, and 3-Cl-Tyr are shown as brown, red, blue, and green dashed lines.
in DMSO;11,12 the ΔpKa series provides a surrogate for charge
accumulation.11
An analogous increased sensitivity due to the active site could
contribute to catalysis. According to this model, as charge
accumulates in the course of a reaction there would be a larger
increase in hydrogen bond energy in the active site relative to
the increase in water.7 However, the energetics of active site
hydrogen bonds cannot be inferred from model compound
studies in any other solvent, because enzyme active sites, with
polar and nonpolar residues that are dynamically restrained and
are surrounded by an aqueous phase, are not analogous to or
authentically modeled by any homogeneous solvent or the gas
phase.13,14 Thus, experimental tests within active site environ-
ments are needed.
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(Louisville, KY, USA); S-trityl-β-mercaptopropionic acid was
purchased from Peptides International (Louisville, KY, USA); and
Boc-Leu-phenylacetamidomethyl resin and 1,4-dithio-DL-threitol
(DTT) were purchased from Bachem (Torrance, CA, USA). All
other N-Boc protected amino acids were purchased from Novabio-
chem (EMD Millipore, Billerica, MA, USA).
5(10)-Estrene-3,17-dione (5(10)-EST) and 4-androstene-3,17-
dione (4-AND) were purchased from Steraloids (Newport, RI,
USA). 5-Androstene-3,17-dione (5-AND) was synthesized from 4-
AND as described by Malhotra and Ringold and purified by silica gel
flash chromatography, eluting with dichloromethane as described by
Pollack et al.50,51 3-Cyclohexen-1-one was synthesized as previously
described.52
KSI Semisynthesis. I. Synthesis of N-Boc, O-Silylated Chloro-
and Fluorotyrosines. Fluorotyrosine Synthesis. Fluorotyrosines were
synthesized on a 20 mmol scale from pyruvic acid, ammonium acetate,
and the corresponding fluorophenol using tyrosine phenol lyase as
previously described.53 Minor modifications described below were
made to the procedure for isolating tyrosine from the reaction mixture
following reaction completion.
After complete conversion of substituted phenol to tyrosine, as
monitored by HPLC, enzyme was precipitated by lowering the
solution pH to 3 by addition of glacial acetic acid (AcOH) and
removed by vacuum filtration through a Celite pad and a 0.2 μm
membrane. Dowex 50WX8 cation exchange resin (100−200 mesh,
100 mL) was washed sequentially with 6 M HCl, 6 M NaOH and
water and then packed into a column and equilibrated with 15%
AcOH in water (v/v). All 2 L of pale yellow filtrate was passed over
this resin using a siphon under gravity, and the resin was washed with
500 mL of 15% AcOH in water (v/v) and with 500 mL of water.
Aqueous ammonia (10% v/v) was used for elution, and fractions
staining positive with ninhydrin were pooled. The resin was washed
with water and then with 15% AcOH in water, and the filtrate was
passed over the resin two more times to maximize amino acid
recovery. Ammonia was removed from the pooled fractions by rotary
evaporation, and any remaining water and other volatile components
were removed by lyophilization to isolate a brown powder. Typical
yields of crude amino acid were 75−90%. This crude product was used
directly for synthesis of the N-Boc protected amino acid without
further purification or characterization.
General Procedure for N-Boc Protection of 3-Chlorotyrosine and
Fluorotyrosines. A 250 mL round-bottom flask equipped with a
magnetic stirbar was charged with 10 mmol of substituted tyrosine
(2.0−2.2 g depending on amino acid molecular weight), 100 mL of 1:1
p-dioxane:water (v/v) and 2.1 mL of triethylamine (15 mmol, 1.5
equiv). The solution was cooled to 0 °C on ice while stirring, and 2.2 g
of di-tert-butyl dicarbonate (11 mmol, 1.1 equiv) was added in one
portion. After stirring on ice for 30 min, the solution was allowed to
warm to room temperature and stirred overnight. Monitoring reaction
progress by thin layer chromatography on silica plates (10% methanol,
0.2% AcOH (v/v) in dichloromethane) revealed the disappearance of
starting material (Rf 0) and the appearance of a spot migrating at Rf
0.4.
Another model for enhanced hydrogen bond contributions
proposes that the enzyme active site environment promotes the
formation of “short, strong” or “low-barrier” hydrogen bonds
by matching the pKa of hydrogen bonding groups in the active
site to those in the transition state.15−19 Indeed, hydrogen
bonds with unusual physical properties, including short donor−
1
acceptor distances, large H NMR chemical shifts, and low
deuterium fractionation factors have been observed in several
proteins.20−28 However, experimental tests of the energetic
significance of such hydrogen bonds have been limited by an
inability to assess the catalytic consequences of systematically
varying the pKa of hydrogen bonding groups while minimizing
accompanying steric or structural changes.29
Computational studies can and have been used to develop
insightful models,30−32 but all calculations employ simplifying
approximations, and theoretical predictions still require
rigorous and independent experimental tests. As noted above,
traditional site-directed mutagenesis studies also do not cleanly
assay the hydrogen bond energetic properties predicted from
the above proposals.
Unnatural amino acids allow maintenance of a hydrogen
bond while perturbing its electrostatic properties and thereby
provide, in principle, a powerful means to probe the energetics
of intact hydrogen bonds within enzyme active sites. Unnatural
amino acids have been incorporated and used in several protein
systems to incisively probe cation−pi interactions, radical
transfer reactions, general acid−base catalysis, hydrogen bonds,
and other molecular properties and interactions.33−39 Here we
use unnatural amino acids to probe the energetics of hydrogen
bonds in the oxyanion hole of bacterial ketosteroid isomerase
(KSI; Figure 1).
KSI, unlike serine proteases, has side chains contributing the
hydrogen bonds within its oxyanion hole and thus allows
systematic perturbation. Prior studies have identified short
hydrogen bonds in the KSI oxyanion hole24,28,29,40−45 and have
proposed that these hydrogen bonds have the energetic
properties described above.16−19,24,40−44,46−49 We report the
catalytic consequences of substituting fluorotyrosines (hereafter
abbreviated as F-Tyr’s; Figure 2A) in place of Tyr16 to
systematically vary the electrostatic properties of this oxyanion
hole hydrogen bond donor. The results provide a test of
whether pKa matching is important for maximal catalysis and a
measure of the sensitivity of catalytic power to the electrostatic
properties of an active site hydrogen bond donor.
EXPERIMENTAL PROCEDURES
■
Materials. All reagents were of the highest purity commercially
available (≥97%). Ultralow total organic carbon (TOC) biological
grade type I deionized water (18.2 MΩ·cm) was generated using an
Aqua Solutions 2121BL system (Jasper, GA, USA) and used to
prepare all buffers and aqueous solutions. Any materials not
mentioned below were purchased from Sigma-Aldrich (St. Louis,
MO, USA), Fisher (Pittsburgh, PA, USA), or Mallinckrodt Baker
(Phillipsburg, NJ, USA).
Dioxane was removed by rotary evaporation, and the remaining
aqueous mixture was cooled on ice. The solution pH was lowered to 2
(measured by pH paper) by the slow, dropwise addition of cold 1 M
HCl while stirring on ice. The acidified mixture was extracted with
ethyl acetate (3 × 20 mL), and the combined organic layers were
washed with brine and dried over anhydrous sodium sulfate. Ethyl
acetate was removed by rotary evaporation to yield a pale yellow oil.
The crude product was dissolved in a minimal volume of
dichloromethane and purified by silica gel flash chromatography
using a gradient of 3−5% methanol in dichloromethane (with 0.2%
AcOH added to lessen product streaking). Solvent was removed by
rotary evaporation and high vacuum to yield a sticky off-white foam
Pyruvic acid (≥98%), p-dioxane (anhydrous, >99.8%) and Dowex
50WX8 cation exchange resin (100−200 mesh) were purchased from
Acros Organics (Geel, Belgium); 3-chloro-L-tyrosine (97%) was
purchased from Alfa Aesar (Ward Hill, MA, USA); di-tert-butyl
dicarbonate (≥99%), trifluoroacetic acid (≥99.9%), O-(benzotriazol-1-
yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (≥99%), N2-
(tert-butoxycarbonyl)-N6-(((2-chlorobenzyl)oxy)carbonyl)-L-lysine
(99%), O-benzyl-N-(tert-butoxycarbonyl)-L-threonine (99%) and N-
(tert-butoxycarbonyl)-L-leucine hydrate (99%) were purchased from
Chem-Impex International (Wood Dale, IL, USA); N2-(tert-butox-
ycarbonyl)-N4-trityl-L-asparagine was purchased from AAPPTec
1
that was characterized by ESI-MS and by H, 13C, and 19F NMR.
Typical isolated yields were 50−80%.
General Procedure for O-Silylation of 3-Chlorotyrosine and
Fluorotyrosines with tert-Butyldimethylsilyl Chloride or tert-
Butyldiphenylsilyl Chloride. This synthesis is based on a previously
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published procedure.54 A 25 mL round-bottom flask, oven-dried,
flushed with argon and equipped with a magnetic stirbar, was charged
with 10 mL of dry N,N-dimethylformamide (DMF), 5 mmol of N-Boc
protected modified tyrosine (1.5−1.6 g depending on amino acid
molecular weight, 1 equiv) and imidazole. Syntheses using tert-
butyldimethylsilyl chloride (TBDMS-Cl) used 3.4 g of imidazole (50
mmol, 10 equiv), while 1.36 g of imidazole (20 mmol, 4 equiv) was
added for syntheses using tert-butyldiphenylsilyl chloride (TBDPS-Cl).
The reaction mixture was cooled to 0 °C on ice, and either
TBDMS-Cl (3.1 g, 20 mmol, 4 equiv) or TBDPS-Cl (1.65 g, 6 mmol,
1.2 equiv) was added in one portion. The solution was stirred at room
temperature under argon for 16 h for reactions using TBDMS-Cl, or
for 36 h for reactions using TBDPS-Cl. Reaction progress was
monitored by TLC on silica gel plates (10% methanol, 0.2% AcOH
(v/v) in dichloromethane), which showed the gradual disappearance
of starting material with Rf 0.4 and the appearance of an intense dark
spot at Rf 0.5.
DMF was removed by rotary evaporation, and the resulting slurry
was dissolved in diethyl ether and washed with 10% citric acid in water
(w/v). The aqueous layers were extracted with diethyl ether (3 × 20
mL), and the combined organic layers were washed with saturated
lithium chloride in water to remove dissolved DMF and dried over
anhydrous sodium sulfate. Diethyl ether was then removed by rotary
evaporation to yield a pale yellow oil.
onto a semipreparative HPLC C18 column (Vydac 218TP510, 5 μm
beads, 250 mm length, 10 mm internal diameter; Grace, Deerfield, IL,
USA) pre-equilibrated with 95% buffer A (water, 0.1% TFA) and 5%
buffer B (9:1 acetonitrile:water v/v, 0.1% TFA) at a flow rate of 1 mL/
min. Gradient elution was then applied at a 5 mL/min flow rate from
95 to 65% buffer A over 20 min, and 65 to 50% buffer A over 30 min.
The identity and purity of the peptide, eluting at a retention time of
46−50 min, was confirmed by ESI-MS. Peptide-containing fractions
were pooled, lyophilized and stored at −80 °C until use. Isolated yields
ranged from 5 to 40 mg (2−15%, theoretical yield = 272−276 mg).
III. Expression and Purification of C-Terminal KSI Polypeptide. A
peptide containing amino acids 24 to 131 of pKSI and incorporating
the mutations D24C/Y32F/C69S/C81S/C97S was isolated and
purified as described in the following subsections. The procedure is
based on one previously described with a few modifications.56 The full
protocol is given below for completeness. All affinity chromatography,
ion exchange and desalting columns used were from GE Healthcare
Life Sciences (Piscataway, NJ, USA) unless otherwise indicated.
Generation of His6-Tagged SUMO−D24C-131/Y32F/C69S/C81S/
C97S KSI Plasmid. QuikChange site-directed mutagenesis (Stratagene,
La Jolla, CA, USA) was used to incorporate the Y32F/C69S/C81S/
C97S mutations into the His6-tagged SUMO−D24C-131 KSI
plasmid.56 Miniprep DNA from DH5α cells was sequenced on an
ABI3100 capillary sequencer (Stanford Protein and Nucleic Acid
sequencing facility).
Expression and Purification of His6-Tagged SUMO-(D24C-131)/
Y32F/C69S/C81S/C97S KSI Fusion Protein. Using the construct
generated above, a fusion protein containing an N-terminal His6 tag,
a small ubiquitin-like modifier protein (SUMO) tag, and the amino
acids 24 to 131 of pKSI with the mutations D24C/Y32F/C69S/C81S/
C97S was expressed in E. coli BL21(DE3) cells grown in 6 L of
lysogeny broth (LB). Cells were grown at 37 °C while shaking at 220
rpm to an OD600 of 0.6−0.8 and then induced with 0.5 mM isopropyl-
β-D-1-thiogalactopyranoside (IPTG) and incubated at 25 °C over-
night. Cells were harvested by centrifugation, resuspended in 80 mL of
cold 20 mM sodium phosphate, pH 7.2, μ = 150 mM NaCl (lysis
buffer), and lysed by passage through an Emulsiflex (Avestin, Ottawa,
ON, Canada). All subsequent steps were performed at 4 °C unless
indicated otherwise.
Inclusion bodies containing fusion protein were isolated by
centrifuging cell lysate at 48400g for 20 min. Inclusion bodies were
then resuspended in 50 mL of lysis buffer containing 1% Triton X-100
using a dounce to remove membrane lipids, centrifuged at 17400g for
20 min at 4 °C, and then washed several times by resuspension in lysis
buffer and centrifugation to remove detergent. Inclusion bodies were
solubilized in 25 mL of 50 mM Tris-HCl, 150 mM NaCl, 20 mM
imidazole, pH 8.0, 7 M urea (binding buffer) using a dounce, followed
by centrifugation at 48400g to remove insoluble material and
clarification by passage through a 0.2 μm syringe filter.
The supernatant was loaded on a 5 mL HisTrap HP column pre-
equilibrated with binding buffer. The column was washed with binding
buffer until no further change was observed in A280. Elution was carried
out using 50 mM Tris-HCl, 150 mM NaCl, 250 mM imidazole, pH
8.0, 7 M urea (elution buffer). The column was then re-equilibrated
with binding buffer, and the flowthrough from the previous run was
passed over the column 5−6 more times with fusion protein being
eluted each time to maximize recovery. All steps were performed at a
flow rate of 4 mL/min.
The crude product was dissolved in a minimal amount of
dichloromethane and purified by silica gel flash chromatography
using a gradient of 2−4% methanol in dichloromethane (with 0.2%
AcOH). Solvent was removed by rotary evaporation and high vacuum
to yield a white foam that was crushed to a white powder,
1
characterized by ESI-MS and by H, 13C, and 19F NMR, and stored
at −20 °C until use in peptide synthesis. Typical isolated yields were
40−60%.
II. Synthesis and Purification of N-Terminal KSI Peptide with a
Thioester for Native Chemical Ligation. N-Terminal Peptide
Synthesis and Cleavage. Peptides containing the first 23 amino
acids of pKSI with the R15K and D21N mutations and with a C-
terminal β-mercaptopropionyl-Leu thioester for ligation were synthe-
sized on 100 μmol scale using Boc-Leu-PAM resin (0.61 mmol/g) in
DMF using manual in situ neutralization protocols as described
elsewhere.55
Tyrosine and its unnatural variants were incorporated at position
16. Tyrosine was incorporated using N-Boc-Tyr-(2-Br-Z)-OH, and 2-
fluorotyrosine was incorporated using N-Boc-O-(tert-butyldimethylsil-
yl)-2-fluorotyrosine, synthesized as described above. 3-Fluorotyrosine,
2,6-difluorotyrosine, and 3-chlorotyrosine were incorporated using the
corresponding N-Boc-O-(tert-butyldiphenylsilyl) amino acids.
Following incorporation of unnatural tyrosine variants into the
peptides, N-Boc deprotection conditions were changed from
incubating the resin in neat trifluoroacetic acid (TFA) twice for 1
min, to incubating in 1:1 TFA:dichloromethane (v/v) twice for 4 min,
with a dichloromethane flow wash before and after N-Boc
deprotection. These conditions were used to minimize premature
removal of the silyl protecting group on the tyrosine hydroxyl before
peptide synthesis was complete.
Peptides were cleaved from the resin by first adding 750 μL of
thioanisole and 5 mL of TFA to dried resin in a 20 mL glass vial while
stirring on ice for 10 min, followed by addition of 500 μL of
trifluoromethanesulfonic acid dropwise and letting the solution warm
to room temperature. After 1 h, the resin was separated from the
solution by filtration through a Kimwipe plug in a glass Pasteur pipet,
and washed with ∼3−4 mL of TFA. The volume of TFA in the filtered
solution was reduced from ∼10 to ∼3−4 mL by evaporation under a
stream of nitrogen. Cold diethyl ether was added slowly while the vial
was vigorously agitated on ice to precipitate crude peptide. The
precipitate was triturated with cold diethyl ether (3 × 15 mL) to
remove any soluble impurities, divided into four portions, dried under
a stream of nitrogen, and stored at −80 °C until purification.
N-Terminal Peptide Purification. Each portion of crude peptide
from above was resuspended in 5 mL of 55% formic acid in water (v/
v), filtered through a Kimwipe plug in a glass Pasteur pipet, and loaded
The eluted material was buffer exchanged into 50 mM Tris-HCl,
150 mM NaCl, pH 8.0, 7 M urea using 10 kDa cutoff Amicon
centrifugal filter units (EMD Millipore, Billerica, MA, USA) to remove
imidazole from the previous step. The eluted material was diluted
while stirring into 50 mM Tris-HCl, 150 mM NaCl, pH 8.0 to a final
urea concentration of 2 M to allow the SUMO protein tag to fold. The
purity of the fusion protein was >95% as assessed by Coomassie-
stained SDS-PAGE.
Cleavage of His6-Tagged SUMO-(D24C-131)/Y32F/C69S/C81S/
C97S KSI Fusion Protein and Purification of D24C-131/Y32F/C69S/
C81S/C97S KSI Polypeptide. The SUMO tag was cleaved by adding
SUMO protease (1 mg protease per 100 mg fusion protein) and DTT
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(2 mM final concentration), and stirring overnight at 4 °C. The
reaction volume was approximately 300 mL, and the concentration of
fusion protein during the cleavage reaction was ∼12 mg/mL on the
basis of a Bradford assay using a KSI-derived standard curve that was
obtained with expressed KSI with its concentration independently
determined by absorbance under denaturing conditions.57 Cleavage
efficiency was >90% as assessed by Coomassie-stained SDS-PAGE.
Solid urea was added to a final concentration of 7 M to solubilize a
small amount of precipitate that was observed at the end of the
cleavage reaction, and imidazole and DTT were added to final
concentrations of 20 mM and 2 mM, respectively. The solution was
clarified by passage through a 0.2 μm membrane under a vacuum.
SUMO protease, SUMO protein tag, and any uncleaved SUMO KSI
fusion protein were then removed by subtractive nickel affinity
chromatography as follows.
The cleavage reaction mixture was passed over a 5 mL HisTrap HP
column pre-equilibrated with 50 mM Tris-HCl, 20 mM imidazole, 150
mM NaCl, 2 mM DTT, pH 8.0, 7 M urea (DTT binding buffer). The
column was washed with DTT binding buffer until no further decrease
in A280 was observed. Elution of bound SUMO tag, protease, and
uncleaved fusion protein was achieved with 50 mM Tris-HCl, 250 mM
imidazole, 150 mM NaCl, 2 mM DTT, pH 8.0, 7 M urea (DTT
elution buffer), and the column was re-equilibrated with DTT binding
buffer. The flowthrough was passed over the column 3 more times to
maximize removal of SUMO tag, protease and uncleaved fusion. Purity
of the cleaved D24C-131/Y32F/C69S/C81S/C97S peptide was
typically >95% as assessed by Coomassie-stained SDS-PAGE.
The cleaved peptide solution was concentrated using 3 kDa cutoff
Amicon centrifugal filter units to a volume of ∼30 mL, buffer
exchanged into 50 mM ammonium bicarbonate by passage through a
HiPrep 26/10 desalting column, and lyophilized to an off-white
powder. Typical yields were ∼150 mg. The lyophilized peptide was
stored at −80 °C until ligation.
IV. Native Chemical Ligation, Folding, and Purification to
Generate Semisynthetic KSI. The synthesized peptide containing
tyrosine variants at position 16 and a C-terminal thioester was ligated
to the recombinant peptide containing an N-terminal cysteine by
native chemical ligation. Lyophilized peptides were added to a final
concentration of ∼2 mM (synthesized peptide) and ∼4−6 mM
(recombinant peptide) to 500 μL of freshly prepared ligation buffer
(100 mM sodium phosphate, 50 mM 4-mercaptophenylacetic acid, 20
mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl), and 6
M guanidine hydrochloride, pH 7.1).
The reaction mixture was incubated at 25 °C for 4 h and then
diluted 200-fold into 40 mM potassium phosphate, 1 mM Na·EDTA,
500 mM L-arginine·HCl, pH 7.2 and stirred at 25 °C for 30 min to fold
ligated material. Folded protein was purified by deoxycholate affinity
chromatography as previously described for other KSI mutants.3
Eluted protein was passed through a 30 mL Superose 12 size exclusion
column pre-equilibrated with 40 mM potassium phosphate, 1 mM
EDTA, 2 mM DTT, for buffer exchange and then concentrated in a 3
kDa cutoff Amicon centrifugal filter unit. Final protein purity was
typically >99% as assessed by Coomassie-stained SDS-PAGE, and ESI-
MS was used to confirm the masses of all semisynthetic proteins.
Typical purified yields relative to the limiting synthesized peptide were
2−5%.
solubility and monitored continuously at 248 nm (5(10)-EST and 5-
AND) or at 240 nm (3-cyclohexen-1-one) in a Lambda 25
spectrophotometer (PerkinElmer, Waltham, MA, USA). Initial rates
of product formation with 5(10)-EST, 5-AND and 3-cyclohexen-1-one
were calculated using previously determined molar extinction
coefficients for the products 4-estrene-3,17-dione (4-EST), 4-
androstene-3,17-dione (4-AND), and 2-cyclohexen-1-one of 14 800,
14 750, and 7680 M−1 cm−1, respectively.52,58 Substrate concentrations
used in the experiments below are summarized in Table S1
(Supporting Information).
Michaelis−Menten Kinetics. Reactions with 5(10)-EST and 5-
AND were conducted in 40 mM potassium phosphate and 1 mM
sodium·EDTA at pH 7.2 as previously described.52 The values of kcat,
KM, and kcat/KM were determined by fitting the initial rates of product
formation as a function of substrate concentration to the Michaelis−
Menten equation. At least three determinations from independent
experiments using different enzyme concentrations varied over 6- to
12-fold were averaged.
Determining kcat/KM values for 3-Cyclohexen-1-one. Reactions
with 3-cyclohexen-1-one were conducted in 4 mM potassium
phosphate, 0.1 mM sodium·EDTA, pH 7.2 buffer as previously
described.52 The substrate concentrations used were chosen on the
basis of prior determinations of KM between 100 and 500 mM for
several pKSI mutants.52 A lower buffer concentration was used with
this substrate than with 5(10)-EST and 5-AND to decrease the
background rate of product formation. Values of kcat/KM were
determined from initial rates. The maintenance of a subsaturating
level of 3-cyclohexen-1-one was tested by ensuring that initial rates
increased by 5-fold upon a 5-fold increase in substrate concentration.
pH-Dependent Activity Measurements. Reactions were conducted
with 5(10)-EST in 50 mM buffer with a constant ionic strength
maintained at 100 mM using NaCl. Buffers with overlapping pH
ranges were used:59 sodium acetate (pH 4.1−5.4), sodium succinate
(pH 4.8−6.4), sodium phosphate (pH 6.2−7.6), sodium N-(tri-
(hydroxymethyl) methyl)glycine (tricine) (pH 7.4−9.1), sodium
glycine (pH 8.9−10.4), and sodium carbonate (pH 9.7−10.8). Activity
measurements were made using initial rates of product formation.
Enzyme concentrations were varied over 3- to 4-fold at each pH to
ensure that reactions were first order in enzyme concentration.
Subsaturating and saturating concentrations of 5(10)-EST were
initially chosen on the basis of prior measurements of KM between
20 and 50 μM for several pKSI mutants,52,60 and similar KM values
were determined in this work from full Michaelis−Menten curves at
pH 7.2.
The observed activity as a function of pH was fit to a model
containing an inactivating deprotonation and protonation (Figure S3A
and S3B, Supporting Information) in Kaleidagraph 4.04 (Synergy
Software, Reading, PA, USA) using eqs 1 and 2 for subsaturating and
saturating activity, respectively.
v0
(
)
⎛
⎞
[E]T[S]T
v0
max
+ 10pH−pK
=
⎜
⎝
⎟
⎠
E,1−pH
E,2
1 + 10pK
[E] [S]
a
a
T
T
(1)
obs
v0
([E] )
⎛
⎞
v0
[E]
T
max
+ 10pH−pK
=
⎜
⎝
⎟
⎠
E·S,1−pH
E·S,2
The concentrations of expressed and semisynthetic enzymes
containing Tyr at position 16 were determined using a calculated
molar extinction coefficient at 280 nm (15 700 M−1 cm−1).57 The
concentrations of semisynthetic enzymes containing unnatural
tyrosines were determined by modifying this calculated extinction
coefficient with the extinction coefficient of the unnatural tyrosine
(data not shown), which was assumed to be the same as that for the
corresponding phenol. Calculating protein concentrations without this
correction (i.e., assuming that the extinction coefficients of tyrosine
and unnatural tyrosines are the same) gives protein concentration
values that are within 8% of the corrected values and has no effect on
the conclusions of this work.
1 + 10pK
a
a
T
(2)
obs
where v0 is the observed initial rate of product formation, [E]T and
[S]T are the total initial concentrations of enzyme and substrate, pKaE,1
and pKaE·S,1 are the pKa values for inactivation at low pH under
subsaturating and saturating conditions, and pKaE,2 and pKaE·S,2 are the
pKa values for inactivation at high pH under subsaturating and
saturating conditions, respectively.
Solvent Isotope Effect Determinations. Reactions were conducted
in 40 mM potassium phosphate, 1 mM sodium·EDTA, pH 7.2 (H2O)
or pD 7.6 (D2O). The pD of deuterated buffer was calculated by
adding 0.4 units to the reading displayed by a pH meter connected to a
glass electrode.61 A deuterated buffer with a pD 0.4 units higher than
the pH of protiated buffer was used to ensure that the same fraction of
Kinetics. All reactions were conducted at 25 °C in buffer
containing 2% DMSO (v/v) added as a cosolvent for substrate
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titratable groups would be ionized in both H2O and D2O reactions, as
the pKa values of most ionizable groups are elevated by 0.4 units in
D2O relative to H2O.61 The reactions in each independent experiment
comparing activity in H2O and D2O for a particular KSI variant were
initiated by adding the same volume of the same concentrated enzyme
stock (prepared in 40 mM potassium phosphate, 1 mM sodium·
EDTA, pH 7.2 (H2O)), to H2O or D2O buffer containing 5(10)-EST.
The final mole fraction of deuterium in the water used in all reactions
conducted in D2O was greater than 99 atom % D.
Solvent isotope effects were determined by dividing the initial rate
of product formation in H2O by the rate observed in D2O (initial rates
were used as deuterium is incorporated into substrate over time; see
Supporting Information Text II). The ratios observed in 6−12
(subsaturating) or 2−3 (saturating) independent experiments were
averaged to obtain a mean and standard deviation for each KSI variant.
Enzyme concentrations were varied over a 3-fold range with 2.3, 4.7,
and 300 μM substrate.
Primary Kinetic Isotope Effect Measurements. Reactions were
conducted in 40 mM potassium phosphate, 1 mM sodium·EDTA, pH
7.2 (H2O) or pD 7.6 (D2O). The pD of deuterated buffer was
determined as described in the section above. Values of kcat/KM were
determined by fitting the change in absorbance with subsaturating
substrate monitored nearly to completion to a single exponential and
plotting the observed rate constants as a function of enzyme
concentration varied over a 3-fold range. Kinetic isotope effects for
the semisynthetic enzymes under subsaturating conditions were
determined by dividing kcat/KM for protiated 5(10)-EST in H2O by
kcat/KM for 4,4-dideuterated 5(10)-EST in D2O. Saturating kinetic
isotope effects were determined by dividing v0/[E] for protiated
5(10)-EST in H2O by v0/[E] for 4,4-dideuterated 5(10)-EST in D2O,
where v0 is the initial rate of product formation observed with 300 μM
substrate.
D2O for 18 min, with either 2% DMSO (150 or 300 μM substrate) or
50% DMSO (7500 μM substrate). Aliquots of these reactions were
diluted 25-fold into deuterated buffer containing semisynthetic enzyme
to start the enzymatic reactions. The activity of semisynthetic enzymes
with protiated 5(10)-EST in H2O was determined using substrate that
had been incubated in H2O instead of 1 mM NaOD/D2O and diluting
into protiated rather than deuterated buffer.
KSI Equilenin Affinity Determinations and Active Site
Titrations. All fluorescence measurements were made at 20 °C on
a FluoroLog-3 spectrofluorometer (Horiba Jobin Yvon, Edison, NJ,
USA) using 45 μL microcuvettes (Starna Cells, Atascadero, CA, USA).
The affinity of D40N-containing KSI variants for EqA488−1, a
fluorescent analogue of equilenin, was determined by fitting the
observed fluorescence of EqA488−1 measured over a range of enzyme
concentrations to a quadratic binding isotherm as described previously
(Table S6, Supporting Information).29 All experiments used 0.1 nM
EqA488−1, and fluorescence measurements were made with no
enzyme and at enzyme concentrations varying in 2-fold increments
from 0.8 to 200 nM. All measurements were conducted in pH 7.2
buffer containing 10 mM potassium phosphate, 0.1 mM sodium·
EDTA and ionic strength maintained at 100 mM using KCl, with
excitation at 480 nm, emission at 515 nm, and excitation and emission
bandpass values of 10 and 14.7 nm, respectively.
Active site titrations were conducted in pH 7.2 buffer containing 10
mM potassium phosphate, 0.1 mM sodium·EDTA, 5% DMSO (v/v)
and ionic strength maintained at 100 mM using KCl, with excitation at
335 nm, emission at 365 nm, and excitation and emission bandpass
values of 2 and 10 nm, respectively. Equilenin fluorescence was
monitored over a range of enzyme concentrations varied up to twice
the concentration of equilenin (900 nM), and the observed data were
fit to the following quadratic binding isotherm using Kaleidagraph 4.04
(Synergy Software, PA):
4,4-Dideuterated 5(10)-EST was generated by incubating 150, 300,
or 7500 μM protiated 5(10)-EST with 1 mM sodium deuteroxide in
F
= Fbound[Eqbound] + Ffree[Eqfree
]
obs
n[ET] + [Eq ] + KdEq
−
(n[ET] + [Eq ] + KdEq)2 − 4n[ET][Eq ]
T
T
T
F
= Fbound
obs
2
−
⎛
⎜
⎞
⎟
⎟
⎠
n[ET] + [Eq ] + KdEq
(n[ET] + [Eq ] + KdEq)2 − 4n[ET][Eq ]
T T
T
+ F [EqT] −
free
⎜
2
⎝
(3)
where n is the fraction of enzyme that is active or can bind equilenin,
KEdq is the affinity for equilenin determined using EqA488−1 in an
independent experiment and held constant, [ET] and [EqT] are the
total concentrations of enzyme and equilenin, respectively, and Fbound
and Ffree are the fluorescence values of the bound and free states.
incorporated into a synthesized peptide fragment containing
the first 23 amino acids of KSI and a C-terminal thioester. The
rest of the protein was expressed as a SUMO fusion, followed
by cleavage and removal of the SUMO tag. Full-length KSI was
produced by native chemical ligation of the two peptides and
folding the ligated material.
Preparation and Initial Characterization of KSI
Variants with Unnatural Tyrosines in the Oxyanion
Hole. Mutants were prepared in the Y32F background, as Y32F
eliminates potentially complicating active site ionizations that
could obscure comparisons of different F-Tyr mutants;56 this
mutant has activity within 2-fold of wild type pKSI (Table S2,
Supporting Information). Mutations R15K and D21N were
also incorporated, as they facilitate peptide synthesis using Boc
chemistry with triflic acid cleavage, and a D24C mutation was
incorporated to facilitate ligation. The surface cysteine residues
C69, C81, and C97 were mutated to serine, as we observed by
ESI-MS that the presence of cysteine residues at those positions
led to the formation of 4-mercaptophenylacetic acid (MPAA)
adducts that could not be disrupted by denaturation or
treatment with DTT (data not shown). Bacterially expressed
RESULTS
■
KSI catalyzes the migration of double bonds adjacent to
carbonyl groups on cyclic substrates (Figure 1). In an initial
enolization step, the general base Asp40 abstracts an α-proton
from bound substrate, generating a dienolate intermediate
stabilized by hydrogen bonding interactions with the side
chains of Tyr16 and Asp103 in an oxyanion hole. In a
subsequent ketonization step, the intermediate is reprotonated
at a different position to generate conjugated product.62
We investigated the catalytic consequences of systematically
varying the electrostatic properties of an oxyanion hole
hydrogen bond donor by substituting F-Tyr’s (Figure 2A) in
place of Tyr16 in the KSI oxyanion hole. F-Tyr substituted KSI
mutants were prepared using a recently published semisynthesis
procedure with modifications described in Experimental
Procedures (Figure 2B).56 F-Tyr’s were protected and
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enzyme containing all of these mutations had activity that
matched that of Y32F KSI within 20%, indicating the absence
of substantial functional effects from these substitutions (Table
S2, Supporting Information). For simplicity, this enzyme is
referred to as WT hereafter.
subsaturating and saturating conditions, respectively (Figure S3,
Supporting Information, Table 2). The numerical agreement
Table 2. pKa Values for Inactivation at High pH for
Semisynthetic KSI Tyr16 Variants from pH Dependencies of
Activity with Subsaturating (pKaE,2) and Saturating (pKaE·S,2
5(10)-EST
)
Semisynthetic WT, 2-F-Tyr, 3-F-Tyr, 2,6-F2-Tyr, and 3-Cl-
Tyr substituted enzymes were folded, purified, and found by
ESI-MS to have the expected masses (Figure 2C). The activity
of semisynthetic WT was 60−80% of that of recombinant WT
enzyme (Figure S1, Supporting Information). The activities of
semisynthetic WT and 2-F-Tyr-substituted enzymes were the
same within 25% across independent preparations (Figures S1
and S2, Supporting Information), suggesting that the activity of
semisynthetic KSI is reproducible between preparations and
that any small systematic differential in activity between
expressed and semisynthetic KSI was also reproducible.
Experiments with the tight-binding inhibitor equilenin, using
semisynthetic KSI also containing a D40N mutation to allow
very strong binding and thus active site titration, gave
stoichiometries near to 1:1 for binding to enzymes with Tyr,
F-Tyr’s, or 3-Cl-Tyr at position 16 (see Supporting Information
Text I, Table S7). These results are consistent with all or nearly
all of each semisynthetic protein being in a fully active
conformation and provide no indication of differential enzyme
misfolding with different unnatural amino acid substitutions.
Semisynthetic KSI pH Dependencies. To compare the
activities of semisynthetic KSIs under reaction conditions
where all variants had the same ionization state, we first
determined pH dependencies of activity under subsaturating
and saturating conditions. We used 5(10)-estrene-3,17-dione
[5(10)-EST] as prior work showed that a chemical step is rate-
limiting for this substrate.63 In all cases, activity was constant
between pH 5 and 9, consistent with all variants having the
same ionization state across this plateau region (Figure S3,
Supporting Information). The measured activities with over-
lapping buffers fall on the same best-fit line, consistent with the
prior observed absence of buffer-specific effects (Figure S3,
Supporting Information).59 Further experiments were carried
out at pH 7.2, in the center of this region. In all variants, an
inactivating protonation, presumably corresponding to proto-
nation of the general base Asp40,64 was observed at low pH and
with the same pKa of 4.3 0.1 (Table 1). This observation is
consistent with an absence of structural or environmental
changes around the general base upon Tyr substitution.
In WT enzyme, an inactivating deprotonation is observed at
high pH with a pKa of 10.00 0.04 and 10.06 0.12 under
a
residue at
position 16
side chain solution
pKaE,2
(subsaturating)
pKaE·S,2
(saturating)
b
pKa
Tyr (WT)
2-F-Tyr
9.95
9.28
8.81
8.35
8.48
10.00 0.04
9.58 0.08
9.84 0.07
9.58 0.04
9.71 0.12
10.06 0.12
10.38 0.07
10.09 0.10
10.28 0.06
9.60 0.08
3-F-Tyr
2,6-F2-Tyr
3-Cl-Tyr
a
Values are from fits to data in Figure S3 (Supporting Information)
and are shown together with standard errors from regression analysis.
b
The side chain solution pKa values are for the corresponding
phenols.86,87
between this value and the Tyr side chain solution pKa 9.95
raised the possibility that this deprotonation corresponds to
ionization of Tyr16. However, similar inactivating pKa values
near 10 were observed for the F-Tyr and 3-Cl-Tyr substituted
KSI variants, despite side chain pKa values in solution that are
considerably lower than 10 (Figure 2A, Figure S3 and S4,
Supporting Information, Table 2). Further, a change in rate-
limiting step is unlikely, given that the same inactivating
deprotonation pKa was observed with a different substrate 5-
androstene-3,17-dione (5-AND) (data not shown). Thus, the
observed constant pKa of 10 likely corresponds to ionization of
a residue other than Tyr16.
Our observations also provide evidence for elevated pKa
values for substituted tyrosines in this position. The solution
pKa values of 9.28, 8.81, and 8.35 for the 2-F-, 3-F-, and 2,6-F2-
Tyr side chains are lower than the observed inactivating pKa
values of 9.6−10.4 (Table 2). For 2,6-F2-Tyr, the most acidic
tyrosine examined in this work, the enzymatic pKa is perturbed
at least 1.23 units relative to its solution pKa value (Table 2).
The unmodified Tyr16 presumably experiences a similar
perturbation (i.e., to ∼11.2 or higher) that is masked by a
different group that titrates with a pKa of around 10 and
inactivates the enzyme.
Dependence of KSI Activity on Tyrosine pKa. To
carefully compare the activities of the F-Tyr-substituted KSI
variants, we determined kcat/KM, kcat and KM at pH 7.2 for
5(10)-EST, a substrate for which prior work has shown that a
chemical step is rate-limiting,63 using nine different substrate
concentrations with 3−4 different enzyme concentrations
(Figure S5, Table S3, Supporting Information). The values of
kcat/KM and kcat showed no significant dependence on tyrosine
acidity, with kcat/KM decreasing by 1.1-fold and kcat by 1.2-fold
over a 40-fold increase in F-Tyr acidity (Figure 3A,B, closed
circles). No change in KM was observed (Figure 3C).
To control for possible complexities with rate-limiting steps
or other idiosyncrasies that might arise with a particular
substrate, we determined the activity of the F-Tyr substituted
KSI variants with two other substrates, 5-androstene-3,17-dione
(5-AND) (Figure S7, Supporting Information, closed circles)
and 3-cyclohexen-1-one, a single-ring substrate (Figure S8,
Supporting Information, closed circles). We found similar
shallow dependencies of activity on F-Tyr pKa. These results
suggest that the previously observed shallow dependence of the
Table 1. pKa Values for Inactivation at Low pH for
Semisynthetic KSI Tyr16 Variants from pH Dependencies of
Activity with Subsaturating (pKaE,1) and Saturating (pKaE·S,1
5(10)-EST
)
a
residue at
position 16
side chain solution
pKaE,1
(subsaturating)
pKaE·S,1
(saturating)
b
pKa
Tyr (WT)
2-F-Tyr
9.95
9.28
8.81
8.35
8.48
4.31 0.05
4.35 0.10
4.30 0.07
4.29 0.05
4.17 0.12
4.28 0.11
4.24 0.07
4.52 0.09
4.18 0.06
4.35 0.06
3-F-Tyr
2,6-F2-Tyr
3-Cl-Tyr
a
Values are from fits to data in Figure S3 (Supporting Information)
and are shown together with standard errors from regression analysis.
b
The side chain solution pKa values are for the corresponding
phenols.86,87
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Figure 3. Dependence of (A) kcat/KM, (B) kcat, and (C) KM for isomerization of 5(10)-EST on the side chain pKa of substituted tyrosine. Tyr and F-
Tyr containing variants are shown as closed circles; 3-Cl-Tyr is shown as open circles. Error bars correspond to standard deviations from 3 to 4
independent experiments using different enzyme concentrations. The slopes in parts A, B, and C are 0.03 0.05, 0.07 0.04, and 0.04 0.04,
respectively.
5-AND reaction on F-Tyr pKa with metabolic F-Tyr
incorporation39 did not arise either from masking of the
chemical step by (partially) rate-limiting binding and release of
5-AND and its product or by modest levels of Tyr
contamination in KSI preparations expressed in the presence
of F-Tyr’s.
Isotope Effects on Semisynthetic KSI Reactions. To
test whether F-Tyr substitutions result in a change in the
mechanism of the KSI-catalyzed reaction, we determined
whether F-Tyr substitutions change the solvent kinetic isotope
effect or the primary or secondary kinetic isotope effects on C−
H abstraction. We observed the same initial rates of product
formation in H2O and D2O with 5(10)-EST (Table 3),
Table 4. Comparison of kcat/KM Values for Isomerization of
Protiated 5(10)-EST in H2O and 4,4-Dideuterated 5(10)-
a
EST in D2O by Semisynthetic KSIs
ratio of kcat/KM
k
cat/KM for
k
cat/KM for 4,4-
values for
protiated 5(10)-
EST in H2O
dideuterated 5(10)- protiated and 4,4-
residue at
position 16
EST in D2O
dideuterated
5(10)-EST
(M−1 s−1
)
(M−1 s−1
)
Tyr (WT)
2-F-Tyr
(8.2 1.6) × 104
(8.3 0.3) × 104
(6.4 1.2) × 104
(6.5 0.8) × 104
(1.2 0.3) × 104
(1.3 0.2) × 104
(7.4 1.2) × 103
(9.5 1.6) × 103
6.8 2.1
6.3 1.0
8.7 2.2
6.8 1.4
3-F-Tyr
2,6-F2-Tyr
a
Conditions: 40 mM potassium phosphate, 1 mM EDTA, 2% DMSO
(v/v), pH 7.2 (H2O) or pD 7.6 (D2O). Values of kcat/KM are from best
fits to data in Figure S13 (Supporting Information) and are shown
together with standard deviations from 3 to 4 independent
experiments using enzyme concentrations varied over a 3-fold range,
using either 6 or 12 μM substrate. Uncertainties in the ratios of kcat/
KM values were obtained by error propagation.
Table 3. Solvent Isotope Effects on the Initial Rates of
Product Formation from 5(10)-EST with Semisynthetic
a
KSIs under Subsaturating and Saturating Conditions
residue at position 16 vH O/vD O (subsaturating) vH O/vD O (saturating)
2
2
2
2
Tyr (WT)
2-F-Tyr
0.99 0.12
1.04 0.13
1.03 0.09
1.09 0.12
0.94 0.05
1.00 0.03
1.10 0.05
1.03 0.01
Table 5. Comparison of kcat Values for Isomerization of
Protiated 5(10)-EST in H2O and 4,4-Dideuterated 5(10)-
a
3-F-Tyr
EST in D2O by Semisynthetic KSIs
2,6-F2-Tyr
a
residue at
position
16
kcat for protiated
kcat for 4,4-dideuterated
Conditions: 40 mM potassium phosphate, 1 mM EDTA, 2% DMSO
5(10)-EST (in H2O)
5(10)-EST (in D2O)
ratio of kcat
values
(s−1
)
(s−1
)
(v/v), pH 7.2 (H2O) or pD 7.6 (D2O). Standard deviations are from 6
to 12 (subsaturating) or 2 to 3 (saturating) independent experiments.
Subsaturating data were collected with 2.3, 4.7, 9.4, 18.8, or 37.5 μM
substrate, and saturating data were collected with 300 μM substrate.
Enzyme concentrations were varied over a 3-fold range with 2.3, 4.7,
and 300 μM substrate.
Tyr (WT)
2-F-Tyr
3.2 0.5
3.1 0.4
2.9 0.6
3.4 0.3
(3.9 0.7) × 10−1
(4.0 0.4) × 10−1
(3.5 0.5) × 10−1
(3.9 0.1) × 10−1
8.1 2.0
7.7 1.2
8.2 2.0
8.9 0.8
3-F-Tyr
2,6-F2-Tyr
a
Conditions: 40 mM potassium phosphate, 1 mM EDTA, 2% DMSO
(v/v), pH 7.2 (H2O) or pD 7.6 (D2O). Values of kcat are from best fits
to data in Figure S14 (Supporting Information) collected at 300 μM
substrate, a concentration shown to be saturating in independent
experiments (Figure S5, Table S3, Supporting Information). Values are
shown together with standard deviations from 2 to 3 independent
experiments using enzyme concentrations varied over a 4-fold range.
Uncertainties in the ratios of kcat values were obtained by error
propagation.
indicating no significant change in the solvent kinetic isotope
effect. We also determined the activity of Tyr and F-Tyr
containing semisynthetic enzymes with protiated 5(10)-EST in
H2O and 4,4-dideuterated 5(10)-EST in D2O (Figures S13 and
S14, Supporting Information; Tables 4 and 5; see Supporting
Information Text II for discussion on the generation of 4,4-
dideuterated 5(10)-EST). Again, there was no significant
change in the ratio of reaction rates of protiated 5(10)-EST
in H2O versus 4,4-dideuterated 5(10)-EST in D2O for the
different enzymes (Tables 4 and 5), suggesting that the primary
and secondary kinetic isotope effects for C−H proton transfer
are also the same for each of these enzymes. These results
suggest that the KSI reaction is unaffected in both rate and
mechanism by F-Tyr substitution. These results also provide no
indication of coupling of proton transfer, which involves Asp40
as a general base, to the electrostatic properties of the oxyanion
hole hydrogen bond donor.65
Assessing Potential Steric Effects. To explore the
potential impact of steric differences between substituted
tyrosines, we measured the activity of KSI with 3-Cl-Tyr.
This side chain has a solution pKa of 8.48, within the pKa range
of the F-Tyr’s examined in this work, but chlorine is larger than
fluorine (covalent radii: Cl = 100 pm, F = 60 pm) and the C−
Cl bond is longer (C−Cl = 174 pm, C−F = 136 pm).66 We
found that kcat and kcat/KM for 5(10)-EST were 4-fold below
the nearly flat line defined by Tyr and the F-Tyr’s (Figure 3,
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open circles). Similar 4- to 7-fold effects were observed with the
two other substrates 5-AND and 3-cyclohexen-1-one (Figure
S7 and Figure S8, Supporting Information, open circles). The
modest but reproducible rate effects for 3-Cl-Tyr and not for F-
Tyr’s suggest that substituents on the tyrosyl ring can affect the
rate of reaction in ways other than perturbing the electrostatic
properties of the hydrogen bond donor.67
substantially greater sensitivity of hydrogen bond energetics to
charge accumulation within the active site than in aqueous
solution, substantial catalysis could accrue via this mechanism.
But determining whether or not there is an enhanced energetic
sensitivity in active site hydrogen bonds, relative to water, i.e.,
determining whether or not this mechanism holds, requires
tests with the enzyme itself, because the enzyme interior cannot
be confidently modeled by the structural or energetic behavior
of any other solvent,13,14 and because its energetic properties
cannot be considered accurately quantitated by current
computational models in the absence of nontrivial predictions
that can be tested by experiment.
The KSI F-Tyr substitutions provide a rare window into this
sensitivity, as the strength of a hydrogen bond donor in the
active site is varied systematically; i.e., among acids of similar
structure, lower pKa acids are stronger hydrogen bond
donors.75 Our observations of nearly flat dependences of
activity on Tyr pKa (Figure 3, Figures S7 and S8, Supporting
Information) suggest that the sensitivity of hydrogen bond
energetics to charge within the KSI oxyanion hole is not as
steep as it is in some organic solvents and is not substantially
steeper than in aqueous solution.76 These results are consistent
with the conclusions of a previous study where little to no
change was observed in the binding affinity of KSI across a
series of fluorophenolate ligands (oxyanion intermediate
analogues) varying in pKa and thereby mimicking charge
accumulation over the course of the reaction.29
The interpretation above assumes that the −F substitutions
affect the hydrogen bond via inductive effects on the tyrosyl
hydroxyl group. But, given the larger size of fluorine relative to
hydrogen, fluorine substitutions on the tyrosyl ring could have
introduced complicating steric effects or structural rearrange-
ments. Nevertheless, the data provide no indication of such
steric effects. All of the F-Tyr’s fall on a single line, along with
the parent Tyr; for the high sensitivity model to hold,
coincidentally larger steric effects for lower pKa F-Tyr’s would
have to precisely compensate for enhanced intrinsic hydrogen
bond strength for each F-Tyr such that the observed straight
line was obtained. Further, although there clearly are steric
effects from the larger chloro-substitution, its deleterious effect
is a modest 4- to 7-fold.
In the absence of “short, strong” pKa-matched hydrogen
bonds or a substantially greater active site sensitivity of
hydrogen bond energetics to charge accumulation in KSI and,
potentially, in other enzymes as well, how can we account for
the enormous rate enhancements attained by KSI and other
enzymes? For KSI, which provides 1012 fold catalysis relative to
a corresponding solution reaction,52 conservative mutation of
the oxyanion hole side chains, Tyr to Phe and Asp to Ala or
Leu give enormous rate decreases of 105−108 fold.68,69 These
observations led to suggestions of distinct structural and
energetic properties of the native hydrogen bonds.19,68,69
However, these mutations create a hydrophobic environment
that is likely destabilizing to the incipient oxyanion relative to
an aqueous environment.3,4
DISCUSSION
■
This study provides tests of proposed models for large catalytic
contributions from active site hydrogen bonds. We first address
the model invoking matched pKa values and then discuss the
model in which the enzyme provides an environment that
substantially enhances the energetic response to charge
accumulation.
Several researchers have proposed that the pKa values or
proton affinities of hydrogen bonding groups in enzyme active
sites are matched to those of the transition state, promoting the
formation of “short, strong” or “low-barrier” hydrogen bonds
that may provide substantial selective transition state
stabilization.15−19 KSI is among the enzymes for which this
proposal has been made,16−19,24,40−44,47 on the basis of H
1
NMR chemical shifts indicative of short oxyanion hole
hydrogen bonds to oxyanionic transition state analogues and
large energetic effects of oxyanion hole mutants.24,41−43,47,68,69
Recently, Childs and Boxer showed that substituted
naphthols bind to KSI in both protonated (neutral) and
deprotonated (anionic) states and observed that the fraction of
bound anionic ligand as a function of napthol ligand solution
pKa fit a titration curve with an equivalence point of 10, similar
to the estimated pKa of the reaction intermediate (Figure 1).70
This observation was regarded as support for catalysis via short,
strong hydrogen bonds that form in the transition state as a
result of matching of proton affinities between the transition
state’s incipient oxyanion and an oxyanion hole hydrogen bond
donor.70
Our observation that perturbation of the Tyr proton affinity
by nearly 2 orders of magnitude (nearly two pKa units, Figure
2A) has <20% effect on the reaction rate, leaving the more than
billion-fold catalysis by KSI virtually intact,52 suggests that there
is no substantial stabilization in this system from the formation
of pKa-matched hydrogen bonds. This conclusion is bolstered
by the expectation that steric effects from the substitutions, if
present, would likely be deleterious, giving a rate decrease in
addition to any from breaking pKa matching. The ability here to
perturb the intrinsic Tyr proton affinity via subtle fluorine
substitutions allows stronger conclusions to be made about the
absence of an energetic contribution from pKa matching,
relative to traditional site-directed mutagenesis in KSI and
other systems.27,71−73
The oxyanion hole Tyr side chain substitutions also test,
more generally, the sensitivity of hydrogen bond energetics to
charge accumulation within the active site environment. Studies
with model hydrogen-bonded complexes have revealed that, in
nonaqueous solvents, the free energy of hydrogen bond
formation is much more sensitive to the electrostatic properties
of the donor and acceptor than in water.7,11 This observation
could be highly relevant to enzymatic catalysis, as the
electrostatic properties of substrates also change as they
morph into transition states and the active site is a “solvent”
environment distinct from aqueous solution.74
Indeed, a different mutational approach by Schwans et al. led
to a much smaller estimate of the contribution from the
oxyanion hole relative to the corresponding reaction in aqueous
solution.60 Schwans et al. replaced the active site hydrogen
bond donors (and surrounding side chains) with a water-filled
cavity and observed a rate decrease of ∼103-fold; several similar
KSI mutants maintained ∼109-fold catalysis relative to the
corresponding solution reaction.60,77 Thus, the KSI oxyanion
Given the electrostatic changes along the KSI reaction
coordinate, and as generally occurs in reactions, if there were
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Article
hole hydrogen bonds appear to contribute ∼103-fold to
catalysis relative to an aqueous environment.
S9); active site titrations of semisynthetic KSI variants (Figure
S10); H NMR spectra of protiated 5(10)-EST incubated with
1
As noted above, our observation of a shallow dependence of
reaction rate over a ∼102-fold change in proton affinity of the
Tyr hydrogen bond donor, and prior observations of little to no
change in the binding affinity of KSI across a series of oxyanion
intermediate analogues varying in proton affinity by ∼102-fold,
strongly suggest that the enzyme environment does not have
energetic properties that substantially enhance the sensitivity of
hydrogen bonds to charge rearrangement relative to that
observed in aqueous solution. Nevertheless, given the large
increase in proton affinity over the course of the reaction,
estimated to be 1013-fold going from the substrate carbonyl to
the intermediate oxyanion,78 the ∼103-fold catalytic contribu-
tion from the oxyanion hole could arise from a slightly higher
energetic sensitivity to charge accumulation within the active
site than in aqueous solution that would not have been detected
in this or the previous study.
However, there are multiple differences between hydrogen
bonds made to water in aqueous solution and those made to
residues in an active site, so that it would be unwarranted to
ascribe the oxyanion hole catalytic contribution solely to a
slightly higher sensitivity to charge. Most basically, positioning
of the oxyanion hole hydrogen bond donors would be expected
to play a role in this catalytic contribution, consistent with
classical descriptions of enzymatic catalysis.81−85 Additional
differences that remain to be dissected and quantitatively
understood include the number of hydrogen bonds formed in
the active site versus water, differences in the identities of the
hydrogen bond donors, the interactions with second solvation
shell water molecules or surrounding enzyme residues, and
differential rearrangements and conformational entropy of
solution versus the enzyme as charge accumulates in going to
the transition state. Such understanding will require systematic
and multifaceted experimental approaches and computation
that provides quantitative and nontrivial predictions that can be
independently tested. The remaining identified catalytic
features, substrate binding and the positioned active site
general base, will also require careful investigation in order to
understand how these and perhaps unidentified catalytic
features provide the remaining 109-fold catalysis.
NaOD/D2O monitored over 18 min, and of 4-EST with no
added base in D2O (Figure S11); product formation from 4,4-
dideuterated 5(10)-EST with semisynthetic WT KSI (Figure
S12); subsaturating activity of semisynthetic KSIs with 4,4-
dideuterated 5(10)-EST in D2O and protiated 5(10)-EST in
H2O (Figure S13); saturating activity of semisynthetic KSIs
with 4,4-dideuterated 5(10)-EST in D2O and protiated 5(10)-
EST in H2O (Figure S14); substrate concentrations used in
kinetic characterizations of KSI variants (Table S1); Michaelis−
Menten parameters for KSI-catalyzed isomerization of 5(10)-
EST by expressed proteins (Table S2); Michaelis−Menten
parameters for isomerization of 5(10)-EST by semisynthetic
KSIs (Table S3); Michaelis−Menten parameters for isomer-
ization of 5-AND by semisynthetic KSIs (Table S4); kcat/KM
values for isomerization of 3-cyclohexen-1-one by semisynthetic
KSIs (Table S5); affinities of expressed and semisynthetic KSI
variants for a fluorescent analogue of equilenin EqA488−1
(Table S6); fractions of expressed and semisynthetic KSI
variants that bind equilenin (Table S7); 1H, 13C, and 19F NMR
spectra and masses of protected tyrosines. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
herschla@stanford.edu
Present Address
†Department of Chemistry and Biochemistry, California State
University Long Beach, Long Beach, California 90840, United
States.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank Tom Muir for suggesting SUMO
fusions as a method to express and purify the C-terminal KSI
fragment, and members of the Herschlag lab for commenting
on the manuscript and for stimulating discussions. This work
was funded by National Science Foundation Grant MCB-
1121778 (to D.H.). A.N. was supported in part by a Howard
Hughes Medical Institute International Student Research
Fellowship, a Natural Sciences and Engineering Research
Council of Canada Postgraduate Scholarship, and a William
and Sara Hart Kimball Stanford Graduate Fellowship. J.P.S. was
supported in part by a National Institutes of Health
Postdoctoral Fellowship.
ASSOCIATED CONTENT
* Supporting Information
■
S
Description of active site titrations of semisynthetic enzymes
(Text I); description of the generation of 4,4-dideuterated
5(10)-EST for kinetic isotope effect determinations (Text II);
comparison of activity of recombinant and semisynthetic WT
KSI with 5(10)-EST (Figure S1); comparison of activity of
independent preparations of semisynthetic 2-F-Tyr KSI with
5(10)-EST (Figure S2); pH dependencies of activity of
semisynthetic KSI variants with 5(10)-EST (Figure S3);
dependence of inactivating deprotonation pKa values observed
in pH dependencies of activity with 5(10)-EST on side chain
pKa of substituted tyrosine (Figure S4); initial rates of product
formation with 5(10)-EST and semisynthetic KSI variants
(Figure S5); initial rates of product formation with 5-AND and
semisynthetic KSI variants (Figure S6); dependence of kcat/KM,
kcat, and KM for isomerization of 5-AND on side chain pKa of
substituted tyrosine (Figure S7); dependence of kcat/KM for
isomerization of 3-cyclohexen-1-one on side chain pKa of
substituted tyrosine (Figure S8); determination of equilenin
affinity for KSI variants containing a D40N mutation (Figure
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