PH dependence of catalysis by pseudomonas aeruginosa isochorismate - Pyruvate lyase: Implications for transition state stabilization and the role of lysine 42

Article abstract of DOI:10.1021/bi200599j

An isochorismate - pyruvate lyase with adventitious chorismate mutase activity from Pseudomonas aerugionsa (PchB) achieves catalysis of both pericyclic reactions in part by the stabilization of reactive conformations and in part by electrostatic transitio

Full text of DOI:10.1021/bi200599j

Article
pubs.acs.org/biochemistry
pH Dependence of Catalysis by Pseudomonas aeruginosa
Isochorismate−Pyruvate Lyase: Implications for Transition State
Stabilization and the Role of Lysine 42
Jose Olucha, Andrew N. Ouellette, Qianyi Luo, and Audrey L. Lamb*
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, United States
ABSTRACT: An isochorismate−pyruvate lyase with adventi-
tious chorismate mutase activity from Pseudomonas aerugionsa
(
PchB) achieves catalysis of both pericyclic reactions in part by
the stabilization of reactive conformations and in part by
electrostatic transition-state stabilization. When the active site
loop Lys42 is mutated to histidine, the enzyme develops a pH
dependence corresponding to a loss of catalytic power upon
deprotonation of the histidine. Structural data indicate that the
change is not due to changes in active site architecture, but
due to the difference in charge at this key site. With loss of the
positive charge on the K42H side chain at high pH, the enzyme
retains lyase activity at ∼100-fold lowered catalytic efficiency but
loses detectable mutase activity. We propose that both substrate
organization and electrostatic transition state stabilization contribute to catalysis. However, the dominant reaction path for catalysis is
dependent on reaction conditions, which influence the electrostatic properties of the enzyme active site amino acid side chains.
he physiological function of the isochorismate−pyruvate
proceed faster than bond formation, or the converse). PchB
is an intertwined dimer with two equivalent active sites, with
each monomer comprising three helices. Each active site is
T
lyase (PchB) from Pseudomonas aeruginosa is to convert
1
4
isochorismate to salicylate and pyruvate. The salicylate is
ultimately incorporated into the siderophore pyochelin from
Pseudomonas aeruginosa for iron scavenging in iron-limiting
environments. PchB cleaves pyruvate from isochorismate with2
quantitative hydrogen transfer from C2 to C9 (Figure 1A),
composed of residues from both monomers, with side chains
derived from the helices and a mobile loop connecting helices
one and two. PchB is a structural homologue of the E. coli
4
chorismate mutase (EcCM) and has nonphysiological
chorismate mutase activity, albeit with considerably lower
1,5
catalytic efficiency than for the lyase activity. The mutase
reaction is a Claisen rearrangement of chorismate to prephenate
in which the pyruvylenol tail is transferred from a C3 ether
6
linkage to a C1−C9 linkage (Figure 1B). Therefore, PchB
catalyzes two pericyclic reactions, which are unusual in biology.
The source of catalytic power for the chorismate mutases is a
matter of debate, and by extension this is true for PchB as well.
Chorismate mutases catalyze the unimolecular rearrangement
of substrate to form product, and the reaction does not involve
an enzyme−substrate covalent intermediate or acid/base
chemistry. Therefore, it has been argued that the enzyme
active site is merely a vessel which orients the pyruvylenol tail
7−11
over the ring such that the reaction is accelerated.
This is
accomplished by the strategic placement of two arginines deep
within the cavity. According to this view, the arginine residues
align the substrate carboxylates, thereby forming a reactive
substrate conformation (or near attack conformation, NAC)
which could provide the driving force for catalysis.
Figure 1. Pericyclic reactions catalyzed by PchB: (A) isochorismate−
pyruvate lyase, a quantitative hydrogen transfer from C2 to C9; (B)
chorismate mutase, a Claisen rearrangement shifting the pyruvylenol
tail from C3 to C1.
and this reaction has been proposed on computational grounds
to be pericyclic. Pericyclic reactions have a transition state that
is cyclic, and bond breaking and formation are concerted,
though not necessarily synchronous (bond breaking may
3
Received: April 19, 2011
Revised: July 8, 2011
Published: July 13, 2011
©
2011 American Chemical Society
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dx.doi.org/10.1021/bi200599j|Biochemistry 2011, 50, 7198−7207

Biochemistry
Article
of salicylate were recorded at pH values from four to nine in
steps of one pH unit for wild-type, K42H-, K42A-, and
K42E-PchB. A multicomponent buffer system was used to
provide a constant ionic strength of 100 mM and to
minimize salt effects: 50 mM formate, 50 mM MES, 100 mM
TrisHCl, and adjusted to the appropriate pH with HCl or
NaOH. All reactions were carried out at room temperature
(25 °C) with 5.3 μM enzyme (wild-type or mutant),
incubated in buffer for 10 min, and initiated with the
addition of 150 μM isochorismate (saturating conditions)
with a total reaction volume of 100 μL. The reactions were
monitored for 50 s by the increase in fluorescence with an
excitation wavelength of 300 nm and emission wavelength of
430 nm using Cary Eclipse fluorescence spectrometer
(Varian). Data were plotted using Kaleidagraph (Synergy
Software).
pH Dependence of the Kinetic Parameters for the
Isochorismate−Pyruvate Lyase Activity (Figure 3).
Kinetic parameters for isochorismate−pyruvate lyase activity
of wild-type and K42H-PchB were determined at pH values
from 3.5 to 9.5 in steps of 0.5 pH units using the formate/
MES/TrisHCl buffer described above. All reactions were
carried out at room temperature with 1.5 μM enzyme (wild-
type or mutant) incubated in buffer for 10 min and initiated
with the addition of 0.1−0.4 mM isochorismate. Product
formation (initial velocity) was linear over the course of the
A counter argument is that the orientation of the substrate in
the enzyme−substrate complex is not a part of the catalytic
picture. Instead, a positively charged amino acid strategically
placed near the ether oxygen of bond breakage provides a
6,12−21
stabilizing effect for the developing negative charge,
and
of course, the arginine−carboxylate interaction would be
present and possibly intensified in the transition state.
Quantum mechanical/molecular mechanical calculations are
in agreement with activation enthalpies and free energies from
experiments and indicate that catalysis is due to a combination
of conformational effects and electrostatic transition state
2
7
1
8,19,22
stabilization.
Mutation of this positively charged active
site amino acid in the chorismate mutases had catastrophic
1
3,15,17,23,24
functional effects.
The reaction that is usually
accelerated by more than a million-fold becomes undetectable.
However, the limited experimental structural evidence (circular
dichroism) suggested that the secondary structure of the
E. coli mutant enzyme was not maintained so some or all of the
loss of catalytic activity could have been caused by loss of
structural integrity rather than loss of the electrostatic
1
7
interaction.
Lys42 of PchB is the only active site amino acid found in the
4
mobile loop between helices one and two and is the positively
charged amino acid comparable to that in the chorismate
mutases hypothesized to be important in electrostatic stabili-
zation of the transition state. Recent work has shown that
mutation at the 42 site does not alter the active site architecture
experiment for all pH values tested. kcat and K were calculated
m
5
in PchB, as evidenced by comparable circular dichroism
using SigmaPlot (SSPS, Inc.) and Kaleidagraph (Synergy
Software).
spectra of WT and K42 mutant enzymes and the X-ray
crystallographic structure of K42A-PchB, which showed
maintenance of active site architecture including the
active site loop. The K42A mutant protein demonstrated a
pH Dependence of the Kinetic Parameters for the
Chorismate Mutase Activity (Figure 4). Kinetic parameters
for chorismate mutase activity of wild-type and K42H-PchB
were determined in the formate/MES/TrisHCl buffer described
above, at pH values ranging from 3.5 to 9.5 in steps of
1
00-fold decrease in both the lyase and mutase activities.
Active site mutations showed covariant changes in kcat/Km
for the two activities, suggesting that the transition states of
the rate-determining steps for both activities are electro-
statically and structurally similar. Unexpectedly, mutational
effects at the active site on kcat showed no correlation
between the two activities, indicating that there are alternate
reaction pathways for the two activities and a structural lack
of similarity between either the reactant or transition states
for the two activities. The importance of the charged amino
acid at the 42 site is further investigated in this work. We
report here the pH profiles for both the mutase and lyase
activities of the wild-type enzyme and of several mutant
forms of the enzyme (K42A, K42H, and K42E) and the
structures of the wild-type and K42E mutant form, both with
the products of the lyase reaction (salicylate and pyruvate)
bound.
0
.5 pH units (however, there was no measurable activity
over pH 8) and pH 7.0 to 8.0 in pH steps of 0.1. Protein
concentrations varied per experiment from 90 to 900 μM
per 100 μL run, with protein concentration increasing
with pH, to optimize the signal-to-noise ratio. Reactions
between pH 3.5 and 7 were carried out at room tempera-
ture. Temperature was kept constant for reactions from
pH 7.0 to 8.0 using a Quantum Northwest TC 125
temperature controller set at 25 °C. PchB wild-type and
PchB K42H were incubated in the buffer for 10 min
before each reaction. Reactions were initiated by the addition
of the chorismate at concentrations ranging from 0.1 to
5
.0 mM and were mixed using a micropipet tip before
placing the cuvette in the spectrophotometer. Initial
velocities were determined by measuring the disappea-
rance of chorismate at 310 nm (ε310 = 370 M− cm ) using
a UV spectrophotometer (Cary 50 Bio, Varian). Substrate
consumption was linear over the course of the experiment
for those pH values that showed detectable activity.
Michaelis−Menten kinetic data were fit by nonlinear
regression. For experimental conditions at pH 7.5 and
above, substrate concentrations were subsaturating; there-
fore, kinetic constants were determined using Henderson
1
−1
MATERIALS AND METHODS
■
Protein Preparation. Wild-type PchB without a histidine
tag was prepared as previously described. K42H-, K42A-, and
K42E-PchB were prepared as previously described.
4
5
Preparation of Isochorismate and Chorismate. Iso-
chorismate was isolated from K. pneumoniae 62-1 harboring the
25
28
entC plasmid pKS3-02 with only minor changes, as described
and Dixon plots. All data were fit using KaleidaGraph
5
previously. Chorismate (Sigma, 60−80% pure) was recrystal-
(Synergy Software) or SigmaPlot (SSPS, Inc.).
26
lized as previously described.
Data Analysis for pH Studies. pH profiles of kcat and
kcat/KM values for substrates were fit to eqs 1-4. Plots for which
the log of the parameter decreased at acidic pH with a slope of
Measurement of Isochorismate−Pyruvate Lyase Ini-
tial Velocities (Figure 2). Initial velocities for the production
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Biochemistry
were fit with eq 1:
Article
1
of 1° oscillations with an exposure time of 20 s and a detector
distance of 290 mm. For K42E, the collected diffraction images
consisted of 1° oscillations with an exposure time of 15 s and a
detector distance of 100 mm. The diffraction data were indexed
29
and scaled using the XDS program package.
(1)
Structure Determination. Structure determination was
Profiles in which the log of the plotted parameter decreased
with a slope of 1 at acidic pH and decreased with a slope of −1
to a lower plateau value at basic pH were fit to eq 2:
performed by molecular replacement for both WT and K42E
30
PchB using Phaser from the CCP4 suite and the PchB
4
pyruvate-bound structure (2H9D) as a model with the water
and ligands omitted. The model building and refinement were
31
32
33
conducted with Coot, Phenix, and REFMAC5.
Crystallographic Models. The final model for wild-type
PchB contains one dimer per asymmetric unit. Monomers A
and B are both composed of residues 2−98. Included in the
model are one salicylate and one pyruvate per active site
(
2)
Profiles in which the log of the plotted parameter decreased at
basic pH with a slope of 2 were fit to eq 3:
(
therefore, two each total) and 44 waters. A Ramachandran plot
34
generated in PROCHECK shows that the model exhibits
good geometry, with 96.5% of the residues in the most favored
regions, 2.3% of the residues in the additionally allowed
regions, and 1.2% in the generously allowed regions. The
residue that is generously allowed is Ala50 of the A monomer
(3)
(
the B monomer has similar ϕ and ψ angles but falls in the
additionally allowed regions). Ala50 of the A monomer is found
in well-defined density and is between two prolines (Pro49-
Ala50-Pro51).
Profiles in which the plotted parameter decreased at acidic pH
with a slope of 1 and decreased at basic pH with a slope of 2
were fit to eq 4:
Table 1. Crystallographic Statistics
WT
K42E
(4)
Data Collection
a
resolution range (Å)
space group
31.71−1.95(2.06−1.95) 25.89−1.79(1.84−1.79)
P22121
P21212
In eqs 1−4, Y is the observed value (kcat or kcat/KM), c is a
pH-independent plateau value of Y, YL is a plateau value of Y
at low pH, YH is a plateau value of Y at high pH, H is the proton
concentration, and Ka1, Ka2, and Ka3 are the apparent
dissociation constants for groups on the enzyme or substrate.
Any data represented as a straight line is a simple linear
regression.
unit cell (Å)
a = 47.3, b = 60.3,
c = 60.6
a = 46.1, b = 57.3,
c = 60.3
observations
unique
12 438
15 294
total
45 612
54 206
completeness (%)
95.6 (97.1)
0.058 (0.226)
15.4 (5.2)
Refinement
97.2 (97.9)
0.098 (0.375)
5.7 (2.0)
b
Rsym
⟨
I/σ(I)⟩
Crystallization of WT and K42E PchB with Salicylate
and Pyruvate. Crystallization was carried out using the
hanging drop vapor diffusion method at 25 °C. Salicylate and
pyruvate were added to purified WT PchB (64 mg/mL) and
K42E PchB (34 mg/mL) such that the ligands were in 20-fold
molar excess, and the solution was incubated on ice for 30 min.
Drops containing 1 μL of the WT/reaction product solution
were mixed with 1 μL of a reservoir solution containing 0.2 M
lithium sulfate, 0.1 M sodium acetate (pH 4.5), and 6%
glycerol. WT parallelepiped crystals grew to ∼0.12 mm ×
resolution range (Å)
31.71−1.95 (2.00−1.95) 25.89−1.79 (1.84−1.79)
no. of reflections
11177 (801)
0.23 (0.26)
0.29 (0.38)
1
13690 (991)
0.21 (0.29)
0.26 (0.41)
1
c
R-factor
d
Rfree
dimers/asymmetric unit
no. of atoms
protein, non-
hydrogen
1570
76
1529
76
non-protein
rms deviations
length (Å)
0
.12 mm × 0.05 mm within 24 h. Drops containing 1 μL of
the K42E/reaction product solution were mixed with 1 μL
of a reservoir solution containing 0.004 M gly, 0.100 M sodium
acetate (pH 3.6), and 12% glycerol. K42E parallelepiped
crystals grew to ∼0.06 mm × 0.06 mm × 0.04 mm in
0.011
1.21
16.3
16.3
14.8
0.022
1.87
20.9
20.6
27.0
angle (deg)
overall B-factor (Å2)
protein
2
days.
salicylate and
pyruvate
Collection of Crystallographic Data. A crystal of WT or
K42E PchB was transferred to a drop of mother liquor
containing 20−32% (v/v) glycerol and flash cooled to −180 °C
with liquid nitrogen. Diffraction data were collected remotely
at beamline 9-2 at the Stanford Synchrotron Radiation
Lightsource. For WT, the collected diffraction images consisted
water
18.3
26.0
a
b
Values in parentheses are for the highest resolution shell. Rsym
=
∑∣I − Iavg∣/∑Iobs where the summation is over all reflections.
obs
c
d
Rfactor = ∑∣F − F ∣/∑F . For calculation of Rfree, 10.0% (WT) and
o
c
o
10.1% (K42E) of the reflections were reserved.
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Table 2. pH Profiles for Wild-Type and Mutant PchB
activity
protein
WT
kinetic parameter (Y)
pK values and kinetic constants
titratable group
isochorismate−pyruvate lyase
kcat
c
0.90 ± 0.01
2.78 ± 0.02
4.17 ± 0.09
2.79 ± 0.08
0.75 ± 0.02
0.03 ± 0.07
3.04 ± 0.00
6.65 ± 0.13
3.40 ± 0.01
1.90 ± 0.00
3.00 ± 0.01
6.29 ± 0.00
2.0 ± 0.1
pKa1
c
isochorismate
isochorismate
kcat/KM
kcat
pKa1
YL
K42H
YH
pKa1
pKa2
YL
isochorismate
His 42
kcat/KM
YH
pKa1
pKa2
c
isochorismate
His 42
chorismate mutase
WT
kcat/KM
kcat
pKa1
c
2.7 ± 0.0
0.6 ± 0.1
chorismate
K42H
pKa1
pKa2
pKa3
c
3.6 ± 0.0
7.7 ± 0.0
8.5 ± 0.1
1.3 ± 0.2
chorismate
His 42
kcat/KM
pKa1
pKa2
9.0 ± 0.6
6.1 ± 0.4
His 42
The final model for K42E-PchB contains one dimer per
asymmetric unit. Monomer A is composed of residues 1−97,
and monomer B has residues 1−41 and 48−97. A portion of
the active site loop is disordered and not built in the B
monomer, which has been documented previously for other
PchB structures. Included in the model are one salicylate and
one pyruvate per active site (therefore, two each total) and 44
waters. A Ramachandran plot generated shows that the model
exhibits good geometry, with 98.8% of the residues in the most
favored regions and 1.2% of the residues in the generously
allowed regions. The generously allowed amino acid is again
Ala50. Data collection and refinement statistics can be found in
Table 1.
range from 4 to 9, with a constant level of activity at pH 5 and
above (within experimental error) as expected based on previous
work. Only the K42H velocity changed significantly as a
5
function of pH in the range of the expected pKa of the side chain.
pH Dependence of Lyase Activity. The effect of pH on
4
,5
the kcat and k /KM values was determined over the pH range
cat
from 3.5 to 9.5 using a buffer system that maintained a constant
ionic strength over the entire pH range. The WT kinetic
parameters were plotted as a function of pH, and the resulting
profile showed a decrease with decreasing pH with a slope of 1
(Figure 3). Fitting of these data to eq 1 indicated one group
with a pKa1 of 2.78 ± 0.02 (k ) or 2.79 ± 0.08 (k /K ) that
cat
cat
M
must be protonated for efficient catalysis (Table 2). We
attribute this pKa1 value to the isochorismate substrate
Structural Analysis. All root-mean-square deviations were
35
36
calculated using LSQMAN in the DEJAVU program package.
(calculated pKa = 3.6 ). K42H was active over the same pH
range. Whereas the wild-type values for kcat and kcat/KM
remained constant at pH values over 4.5, the kinetic parameters
for K42H peaked at pH 5 and then decreased until pH 7, at
which point they maintained a constant value until pH 9. These
data were fit to eq 2 and indicated a comparable pKa1 of 3.04 ±
Protein Data Bank Submission. The atomic coordinates
and structure factors (entries 3REM and 3RET) have been
deposited in the Protein Data Bank, Research Collaboratory
for Structural Bioinformatics, Rutgers University, New
Brunswick, NJ.
0
.00 (kcat) or 3.00 ± 0.01 (kcat/KM) for isochorismate. A
‡
Calculation of ΔΔG . The “mutational effect” was
second ionizable group must be protonated for most efficient
catalysis and displays a slope of −1 between pH 6 and 7. We
attribute this to the histidine of the active site K42H mutation,
since this is not evident in the WT protein (PchB contains one
naturally occurring histidine which is on the protein surface).
determined by dividing k /K value or k value of the
cat
m
cat
5
mutant enzyme by that of the wild type, as done previously.
The ΔΔG was calculated from the equation
‡
pKa2 values calculated for this group are 6.65 ± 0.13 (k ) or
cat
6
.29 ± 0.00 (k /K ). Therefore, the positive charge at the 42
cat M
position significantly promotes catalysis: ∼5-fold difference in
kcat and greater than a 100-fold difference in kcat/KM at high pH
between WT and K42H when the histidine is not protonated.
Nevertheless, there is a significant contribution to catalysis
unrelated to the positive charge allowing for lyase activity when
the active site histidine is not protonated.
pH Dependence of Mutase Activity. WT was active
across the entire pH range from 3.5 to 9.5. The WT mutase
kinetic parameters were plotted as a function of pH, and the
resulting profile was fit by a simple linear regression (kcat) or as
RESULTS
■
Initial Velocity Data of WT and Mutant PchB. As an
initial experiment, the isochorismate−pyruvate lyase activity
of wild-type and mutant PchB (K42A, K42E, and K42H)
was determined by monitoring the accumulation of the product
salicylate by fluorescence over a range of pH values. The initial
velocities at saturating isochorismate concentrations are plotted
versus pH (Figure 2). K42E displayed no detectable activity at
any pH tested. WT and K42A were active across the entire pH
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Biochemistry
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3
6
for the lyase reaction with eq 1 due to a decrease with
decreasing pH with a slope of 1 (kcat/KM) (Figure 4). Fitting of
the k /K data indicated one group with a pKa1 of 2.7 ± 0.0
substrate (calculated pK = 3.6 ). The K42H mutase activity
a
was limited by our ability to accurately measure such a slow
reaction. Unlike the lyase activity, K42H had no detectable
mutase activity above pH 7.8. The kcat for K42H peaked at pH
5.5; indeed, the mutant enzyme was more active than WT in
this pH range and then decreased until pH 7.8. The mutase kcat
data were fit to eq 4, where kcat decreases at low pH with a
slope of 1 and decreases at around pH 7 with a slope of −2.
This fit indicated a comparable pKa1 of 3.6 ± 0.00 (kcat) for
chorismate and two enzymatic ionizable groups that eliminate
activity when deprotonated with a pKa2 value of 7.7 ± 0.0,
which is presumably the K42H side chain, and also a pKa3 value
of 8.5 ± 0.1. The kcat/KM−pH profile for the K42H mutase
activity was best fit to an equation for two ionizable groups at
neutral pH (eq 3). This fit indicated two enzymatic ionizable
groups that eliminate activity when deprotonated with a pKa1
value of 9.0 ± 0.6 and a pKa2 value of 6.1 ± 0.4 which we
attribute to the K42H side chain. There was no detectable
activity at pH values above 7.8, which may indicate that the
positive charge at the 42 position is important for the mutase
activity or may represent the limit of our ability to detect activity.
cat
M
(
(
kcat/KM) that must be deprotonated for efficient catalysis
Table 2). We attribute this pKa1 value to the chorismate
Figure 2. pH dependence of the initial velocities of the lyase activity
for wild-type (◻), K42H (○), K42A (◇), and K42E (×). All
reactions were carried out at room temperature (22 °C) with 5.3 μM
enzyme (wild-type or mutant) and initiated by the addition of 150 μM
isochorismate.
Structural Information for WT and K42E PchB.
Circular dichroism spectroscopy has been used previously to
assess the secondary structure of PchB. Spectra for the three
mutant forms of PchB tested in this work were found to
Figure 3. Dependence of lyase kinetic parameters on pH. (A) kcat−pH profiles and (B) kcat/Km−pH profiles for WT (◻) and K42H (○). The WT
data were fit to eq 1 (solid line) and for K42H to eq 2 (dashed line).
Figure 4. Dependence of mutase kinetic parameters on pH. (A) kcat−pH profiles for WT (◻) and K42H (○). The WT data were fit to a simple
linear regression (solid line) and for K42H to eq 4 (dashed line). (B) kcat/Km−pH profiles for WT (◻) and K42H (○). The WT data were fit to
eq 1 (solid line) and for K42H to eq 3 (dashed line).
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Figure 5. WT and K42E structures. (A) Cartoon of the WT (gray) and K42E-PchB (purple) active sites superimposed highlighting the conservation
of secondary structure and active site loop conformations. Salicylate and pyruvate are shown as sticks, along with the active site residues displayed in
panel C. (B) 2Fo − Fc electron density map of the K42E active site contoured at 1.5 σ. The mutated residue is shown in yellow, the salicylate in gray,
and the pyruvate in pink. (C) Close-up of the active site overlay in panel A (with cartoon removed for clarity) which shows the maintenance of
41
conformation of the active site side chains and the ligands. Figure generated using PyMol.
be comparable to wild-type with strong minima at 208 and
negative side chain (K42E) led to abrogation of detectable
activity. We have also shown that the K42H mutation had
5
5
2
12 nm, characteristic of helical structure. Circular dichroism
spectra at the extreme pH values (3.5 and 9.5) gave traces
comparable to those previously reported (data not shown). WT
PchB and mutants also crystallize into comparable structures
differing kinetic constants at pH 5 and pH 7.5. Therefore, we
proposed that study of this functional PchB mutant with a
titratable charge could provide insight into the alternate
reaction pathways of the pericyclic reactions catalyzed by the
enzyme. Both reactions in the wild-type active site generate
essentially pH-independent values of kcat and kcat/Km between
pH 5 and 9.5 (Figures 3 and 4). This is consistent with both the
reactive substrate conformation and electrostatic transition state
4,5
from conditions ranging in pH values from 3.5 to 8.5. Two
new structures are reported here: the WT structure with the
products of the lyase reaction (salicylate and pyruvate) bound
and the K42E structure with the same ligands (Figure 5). The
K42A structure with the same ligands has been reported
5
previously. The K42H protein crystallized and diffracted to
stabilization hypotheses because the pK of Lys42 may be >10.
a
́
better than 3 Å resolution; however, the data were insufficient
However, when Lys42 is mutated to histidine with an expected
for structure solution (too highly mosaic) despite repeated
trials. The root-mean-square deviations for all of these
structures range from 0.42 to 0.66 Å for 96−97 of the 101 α-
carbons, indicating the very high conservation of fold. A stereo
image of the active site is displayed in Figure 5C to further
emphasize that the active site of the K42E mutant enzyme and
WT overlay precisely and that the loss of activity in this mutant
is not the result of changes in the active site architecture.
pK of ∼6, the values of all rate constants are somewhat
a
reduced (with the exception of the k for the mutase activity
cat
which is actually greater at low pH) and develop a pH
dependence corresponding to a loss of catalytic power upon
deprotonation of the histidine. Furthermore, any difference in
activity between the unprotonated histidine and the alanine
mutant forms may be due to a difference in hydrogen bonding
from the amino acid at this site and the substrate: hydrogen
bonding between a lysine or histidine at this site may promote
loop closure and formation of the proper substrate conforma-
tion for catalysis which is missing in the alanine mutant form.
DISCUSSION
■
Rationale of the K42H Mutant. We have previously
shown that mutation of the amino acid at the 42 site in PchB to
a neutral side chain (K42Q, K42A) caused ≥100-fold decrease
in both mutase and lyase activity, whereas mutation to a
Lyase Activity. For the physiological lyase activity, the
results are straightforward. Both WT and K42H demonstrated
Michaelis−Menten kinetics over the entire pH range tested
7
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dx.doi.org/10.1021/bi200599j|Biochemistry 2011, 50, 7198−7207

Biochemistry
3.5−9.5). k is titrated with pH, with an increase in the
Article
‡
(
ΔΔG of 3.4 kcal/mol resulting from contributions due to the
loss of the positive charge and from an alteration in shape of
the active site loop to accommodate the Lys to His change.
Therefore, the loss of the positive charge at the 42 site
contributes 2.4 kcal/mol (3.4 kcal/mol − 1.0 kcal/mol) and
represents an experimentally determined free energy contribu-
tion from electrostatic transition state stabilization for the lyase
activity. We would like to note that we have previously reported
cat
catalytic rate as the substrate isochorismate is deprotonated
pH 3.5−5). For only the mutant protein, there is a decrease in
rate with the deprotonation of the mutant histidine (pH 5.5−
.5). This deprotonation event has a characteristic pKa for the
(
7
histidine side chain (∼6.5) and a slope of −1, indicative of a
change in a single ionizable group. Once a pH of 8 is reached,
the catalytic rate again becomes stable. Over the pH range
tested, the wild-type enzyme had a stable Km, whereas the Km
of the K42H protein gradually increased. For K42H, the effects
on kcat and Km over the pH range are comparable with about an
order of magnitude change between the most active pH and the
least.
‡
a mutational effect of 0.01 for the K42A mutation (ΔΔG of
2
.7 kcal/mol), which is in good agreement with the calculations
here, and that the K42A-PchB structure showed a conservation
5
of active site architecture.
The same calculations can be made relative to kcat (Table 3).
The reactant state for these calculations is the enzyme−
substrate complex, which in principle could be either stabilized
or destabilized by the electrostatic charge between the wild-type
and K42H mutant. At pH 5.5, the kcat mutational effect was 0.8,
Contribution of Positive Charge at 42 Site to Lyase
Activity. A valuable method for measuring ΔΔG for the
enzyme substrate complex is the determine the K for the
d
substrate. This approach provides information for reactions in
which the ensemble of the ES complex for substrate binding is
‡
which represents a ΔΔG of 0.13 kcal/mol, and at pH 9, the
‡
equivalent to that for the transition to the ES . This is valid for
‡
kcat mutational effect is 0.2, which represents a ΔΔG of
many systems but not for PchB, which is hypothesized to have
a change in conformation (loop closure) between the ES and
0
4
.95 kcal/mol. Therefore, the loss of the positive charge at the
2 site contributes 0.82 kcal/mol (0.95 kcal/mol − 0.13 kcal/
‡
ES and for which loop dynamics is proposed to be important
mol) and represents a net free energy contribution for
electrostatic transition state stabilization of the lyase activity
from the enzyme−substrate complex to the transition state. In
other words, this is the mutational change in free energy for the
transition state corrected for the mutational change in the free
energy of the reactant state (the enzyme−substrate complex).
3
7
in catalysis. However, we can easily measure k /K and k
cat
m
cat
to provide an alternate approach for determining the needed
information for the ensemble ES substates involved in the
catalytic cycle. A mutation may affect either or both of the two
kinetic parameters k /Km and k . A mutational effect on
cat
cat
k /K reports on mutational changes in the free energy of the
cat
m
‡
Since the ΔΔG of the transition state between the wild-type
and K42H mutant remains constant between the two
calculations (2.4 kcal/mol), the enzyme−substrate complex
the reactant state of the kcat calculations) in the K42H mutant
catalytic transition state versus mutational effects on the free
energy of the kcat/Km reactant state: free enzyme and free
substrate. A mutational effect on kcat reports on mutational
changes in the free energy of the catalytic transition state versus
mutational changes in the free energy of the reactant state for
kcat, which is the enzyme−substrate complex.
We propose that the mutation from lysine to histidine is
likely to have no substantial effect on the free energy of the free
enzyme, since changes in the free energy would probably have
structural consequences. Although we cannot derive the free
energies from the structural observations, the extremely similar
circular dichroism spectra and crystallographic structures for
the wild-type and mutant enzymes illustrate that the active site
architecture is conserved. Therefore, we argue that the free
energies of the reactant states for kcat/Km (free enzyme) are
relatively unchanged between wild-type and the K42H mutant.
(
must be stabilized by roughly 1.6 kcal/mol relative to WT
(
2.4 kcal/mol − 0.82 kcal/mol).
Mutase Activity. Analysis of the chorismate mutase
activity is less straightforward: since there is no plateau at
high pH, we cannot do a full analysis. If the lyase and mutase
activities were covariant with respect to k , then we would
cat
expect a kcat of 0.3 min− at pH 9 for the mutase reaction
1
(
Figure 4A), which is well above our limit of detection.
5
However, in agreement with our previous data, the two
activities are not covariant for kcat, with differently shaped curves
and no detectable mutase activity above pH 7.8. Nevertheless,
the pH dependency of the mutase reaction for K42H kcat can be
fit to a curve representing the deprotonation of the substrate
from pH 3.5 to 5.5 (increasing k ) and the deprotonation of
the histidine and a second enzyme-derived ionizable group from
pH 5.5 to 7.5 (decreasing k ). Interestingly, from pH 4.5 to 6,
Calculations of the mutational effect at this site using k /Km
cat
yield the contribution of electrostatics for the 42 site. At pH
cat
5
.5, where K42H-PchB is most efficient, the kcat/Km mutational
‡
effect was 0.2, which represents a ΔΔG of 1.0 kcal/mol, most
likely the effect relative to side chain shape and thus the
position of the positive charge in the active site (Table 3). At
pH 9, the kcat/Km mutational effect is 0.003, which represents a
cat
the K42H has a larger kcat than the wild-type enzyme.
Contribution of Enzyme-Derived Ionizable Groups to
Mutase Activity. The lyase and mutase covariance in kcat/Km
Table 3. Contribution of Positive Charge at 42 Site to Lyase Activity
mutational
effect
(mutant/WT)
mutational
effect
(mutant/WT)
ΔΔG‡
(kcal/mol)
ΔΔG‡
(kcal/mol)
kcat/KM (M− s−1
1
)
kcat (min−1
)
free energy contribution
pH
9
WT
16300 ± 700
47 ± 4
6.5 ± 0.4
1.3 ± 0.1
two contributions from difference in
protonated lysine versus unproto-
nated histidine (steric and charge)
K42H
0.003
0.18
3.4
0.2
0.8
0.95
5.5
WT
11800 ± 100
2100 ± 100
9.2 ± 0.2
7.1 ± 0.4
one contribution from difference in
protonated lysine and protonated
histidine (steric only)
K42H
1
0.13
0.82
2
.4
subtraction of above contributions
leaving only charge contribution
7
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dx.doi.org/10.1021/bi200599j|Biochemistry 2011, 50, 7198−7207

Biochemistry
Article
5
shown previously was not borne out by these pH profiles, with
the caveat that we cannot determine kcat/Km for an enzyme that
does not turnover detectably. It is tempting to hypothesize that
the positive charge is required for mutase catalysis such that
when the histidine is deprotonated the protein is no longer a
catalyst. However, this hypothesis is confounded by two
problems. First, the data could merely be limited by our ability
to measure the very slow mutase reaction in the mutant enzyme
above pH 7.8. Second, the curves fit in Figure 4 indicate there is
a deprotonation of two ionizable groups. We assign one of these
groups to the histidine at the mutated 42 site, in agreement
with the lyase activity. The second ionizable group is also
assumed to be enzyme-derived, and it is possible that this
second ionization has an amplified effect on the poor mutase
activity. There are two possibilities (that we can think of) for
this ionizable group. First, within the active site, there are two
arginines that charge-pair to the carboxylates of the substrate.
If one of these arginines were to be deprotonated, then the
substrate would not be as well-oriented in the active site,
herein using the K42H mutant is approximately a third of the
free energy difference between the PchB-catalyzed and
39
uncatalyzed lyase reactions (7.43 kcal/mol). This suggests
that substrate preorganization is the more energetically costly
of the two contributing factors. Of note, there is a considerable
entropic penalty for transition from the enzyme−substrate
complex to the transition state for this reaction. However, the
entropic penalty has been argued to be compounded by the
organization of the enzyme during active site loop closure,
which may occur after the formation of the enzyme−substrate
39
complex and concomitantly with substrate organization.
Conclusions. If the k /Km is covariant for the two
cat
activities over the pH range, then the pH dependence reflects
the behavior of the free enzyme with both substrates. In other
words, the transition states of the rate-determining steps for
the lyase and mutase activities should be structurally and
electrostatically similar. However, we cannot detect activity in
the K42H mutant above pH 7.8, making it impossible to
determine the kcat/Km at high pH for the mutase activity of the
mutant enzyme. There are at least two reasons to believe that
kcat/Km is not covariant for the two activities over the pH range.
First, the transition state of the rate-determining step cannot
possibly be electrostatically similar at low and high pH for
the K42H mutation if the residue at the 42 site is involved in
catalysis. Second, the curves in Figures 3B and 4B are clearly
not the same shape, even ignoring our inability to measure
activity at high pH for the mutant. Together, this may suggest a
different level of contribution of this charged amino acid in the
two activities.
thereby slowing catalysis. However, the measured pK values
a
(
8.5−9) would indicate a significant change from that of
arginine in solution (12.5) and should reasonably also cause a
similar effect on the lyase activity. A second, more favored
hypothesis is that there is an ionizable group that is involved in
protein structure stability. When this group is deprotonated, it
is possible that a hydrogen bond or salt link is lost (or formed)
that promotes enzyme stability (or flexibility), thereby slowing
catalysis. While this change may be small and not evident in the
robust lyase activity, a small change in structural stability or
flexibility could play a major role in loss of the poor mutase
activity.
40
Benkovic and colleagues have proposed the “catalytic
network” that provides for the ensemble conformation of a
protein as a third dimension to the standard reaction coordinate.
In this hypothesis, a reaction coordinate becomes a three-
dimensional “mountain range” and the authors note that reaction
conditions, such as pH, may alter the dominant path from
Previous pH Dependence Studies on This Enzyme.
Combinatorial mutagenesis experiments of PchB identified
38
Gln90 as an important residue in catalysis. This residue
4
hydrogen bonds to the substrate/product and is within
40
substrate to product. Since the reactant state for kcat/Km is the
free enzyme, and our structural data suggest a conservation of
the architecture of the reactant state, we propose that the path
through the catalytic network is altered with changing pH. In
the case of the lyase activity, a path from reactants to products
is accessible at all of the values of pH tested; however, the
efficiency of the reaction varies depending on the whether or not
a positive charge is present to stabilize bond breaking at the ether
oxygen. For the mutase activity, there may be no accessible path
hydrogen bonding distance to Lys42. Q90E activity was titrated
by changing the pH: the enzyme was more efficient at low pH
(
kcat/Km) when the Glu was protonated and could thus interact
38
with substrate. Interestingly, Q90E showed covariance for the
two activities over the pH range tested,
5−9
and changes in
catalytic efficiency were due to changes in Km (kcat remained
constant). This led to the conclusion that substrate binding is
dependent on protonation of the glutamic acid, which mimics
the naturally occurring glutamine at low pH. These data are
consistent with EcCM, in which the homologous mutation
(
no conformation in the sampled ensemble that promotes
catalysis) without electrostatic transition state stabilization.
17,24
could be rescued at low pH.
We propose an alternative
hypothesis: since Q90E is within hydrogen-bonding distance of
Lys42, the previously reported change in catalytic efficiency may
be an effect of a direct interaction between the two amino acids
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (785) 864-5075. Fax: (785) 864-5294. E-mail: lamb@
ku.edu.
(Q90E and K42) at higher pH. This interaction would promote
closing of the active site without substrate, change the Km, and
not be related to the catalytic mechanism.
Funding
Enzyme Catalysis. Formation of a reactive substrate con-
formation and electrostatic transition state stabilization have
been argued to contribute equally to catalysis, accounting for
the 9.1 kcal/mol difference between the B. subtilis chorismate
This publication was made possible by funds from the
American Lung Association of Kansas and from the Kansas
Masonic Cancer Research Institute, by the Graduate Training
Program in Dynamic Aspects of Chemical Biology NIH grant
T32 GM08545 (J.O.) from the National Institute of General
Medical Sciences, by NIH grant P20 RR016475 from the
INBRE Program of the National Center for Research
Resources, and by NIH grants R01 AI77725 and K02
AI093675 from the National Institute for Allergy and Infectious
Disease.
12
mutase catalyzed and uncatalyzed mutase reactions: the
‡
ΔΔG calculated by quantum mechanics/molecular modeling
methods for the electrostatic transition state stabilization was
1
2
4
.2 kcal/mol and for the free energy cost of substrate pre-
18,20
‡
organization was 3.8−5 kcal/mol.
mol for electrostatic transition state stabilization calculated
The ΔΔG of 2.4 kcal/
7
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dx.doi.org/10.1021/bi200599j|Biochemistry 2011, 50, 7198−7207

Biochemistry
Article
(
14) Kast, P., Asif-Ullah, M., and Hilvert, D. (1996) Is chorismate
ACKNOWLEDGMENTS
■
mutase a prototypic entropy trap? -- activation parameters for the
Bacillus subtilis enzyme. Tetrahedron Lett. 37, 2691−1694.
Diffraction data were collected at the Stanford Synchrotron
Radiation Laboratory, a national user facility operated by
Stanford University on behalf of the U.S. Department of
Energy, Office of Basic Energy Sciences. The SSRL Structural
Molecular Biology Program is supported by the Department of
Energy, Office of Biological and Environmental Research, and
by the National Institutes of Health, National Center for
Research Resources, Biomedical Technology Program, and the
National Institute of General Medical Sciences. We thank the
staff at the Stanford Synchrotron Radiation Laboratory for their
support and assistance. We are grateful to Drs. T.C. Gamblin
and R. N. De Guzman for equipment use and to Dr. R. L.
Schowen for insightful discussions.
(15) Kast, P., Asif-Ullah, M., Jiang, N., and Hilvert, D. (1996)
Exploring the active site of chorismate mutase by combinatorial
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ABBREVIATIONS
PchB, isochorismate−pyruvate lyase from Pseudomonas aeruginosa;
EcCM, chorismate mutase from E. coli.
■
Hermann, J. C., and Mulholland, A. J. (2004) Transition state
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