Entropic and enthalpic components of catalysis in the mutase and lyase activities of pseudomonas aeruginosa PchB

Article abstract of DOI:10.1021/ja202091a

The isochorismate-pyruvate lyase from Pseudomonas aeruginosa (PchB) catalyzes two pericyclic reactions, demonstrating the eponymous activity and also chorismate mutase activity. The thermodynamic parameters for these enzyme-catalyzed activities, as well a

Full text of DOI:10.1021/ja202091a

ARTICLE
pubs.acs.org/JACS
Entropic and Enthalpic Components of Catalysis in the Mutase
and Lyase Activities of Pseudomonas aeruginosa PchB
Qianyi Luo, Kathleen M. Meneely, and Audrey L. Lamb*
Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, United States
ABSTRACT: The isochorismate-pyruvate lyase from Pseudo-
monas aeruginosa (PchB) catalyzes two pericyclic reactions,
demonstrating the eponymous activity and also chorismate
mutase activity. The thermodynamic parameters for these
enzyme-catalyzed activities, as well as the uncatalyzed isochor-
ismate decomposition, are reported from temperature dependence of kcat and kuncat data. The entropic effects do not contribute to
enzyme catalysis as expected from previously reported chorismate mutase data. Indeed, an entropic penalty for the enzyme-
catalyzed mutase reaction (ΔSq = ꢀ12.1 ( 0.6 cal/(mol K)) is comparable to that of the previously reported uncatalyzed reaction,
whereas that of the enzyme-catalyzed lyase reaction (ΔSq = ꢀ24.3 ( 0.2 cal/(mol K)) is larger than that of the uncatalyzed lyase
reaction (ꢀ15.77 ( 0.02 cal/(mol K)) documented here. With the assumption that chemistry is rate-limiting, we propose that a
reactive substrate conformation is formed upon loop closure of the active site and that ordering of the loop contributes to the
entropic penalty for converting the enzyme substrate complex to the transition state.
’ INTRODUCTION
and Streptomyces aureofaciens (SaCM), as the original work was
completed on enzyme purified from bacterial culture.14 A BLAST15
search reveals that K. pneumoniae has a prephenate dehydratase
analogous to EcCM, and an alignment reveals that the CM domains
share 92% sequence identity (all sequence comparisons were
calculated using L-ALIGN16). Early work suggested two CM active
sites,14 so we will classify this enzyme as a member of the AroQ
family. S. aureofaciens, in contrast, contains a monofunctional choris-
mate mutase that shares sequence homology to BsCM (38%
identity, 64% similarity). SaCM was originally thought to have
two or four subunits per oligomer,14 but in recent work, the
calculations have assumed trimeric enzyme.17 We will classify this
enzyme has a member or the AroH family.
The isochorismate-pyruvate lyase (PchB) from Pseudomonas
aeruginosa catalyzes an asynchronous [1,5]-sigmatropic shift with
a hydrogen transfer from C2 to C9 that has been shown to have a
pericyclic transition state: the enzyme-catalyzed reaction shows
quantitative transfer of the C2 hydrogen and a 2H kinetic isotope
effect consistent with the elimination reaction1 (Figure 1A). PchB
also exhibits chorismate mutase (CM) activity (Figure 1B), which is
a pericyclic reaction, albeit more than 2 orders of magnitude less
efficient than physiological chorismate mutases.2,3 Therefore, this
enzyme performs two related pericyclic reactions in a single active
site with a considerable difference in efficiency.
PchB is neither structurally nor functionally homologous
to the pyruvate lyases of other shikimic acid metabolites.4ꢀ6
Instead, PchB shares homology with the chorismate mutases of
the AroQ structural class, including Escherichia coli chorismate
mutase (EcCM).7 EcCM is actually the N-terminal domain of a
two-domain multifunctional prephenate dehydratase that was
cloned separately and purified.8 The structure of EcCM reveals an
intertwined dimer of three helices each with two equivalent buried
active sites composed of residues from each subunit.9 A transition
state analogue (TSA) is bound in the active site, oriented by arginine
residues that align the carboxylates. In contrast, the chorismate
mutase from Bacillus subtilis (BsCM) is a member of the AroH
structural class, and is a trimer which forms a pseudo-Rβ-barrel with
three equivalent active sites at the subunit interfaces. The same TSA
is bound in one of the solved structures, and is oriented in the active
site by comparable arginines.10,11 Apo- and prephenate-bound
structures of BsCM have also been completed.10,12 Despite the
overall structural differences between the two classes, the active sites
of EcCM and BsCM are usually considered comparable due to their
shape and charge complementarity.13
There has been the assertion that the enzyme-catalyzed
chorismate mutase reaction is entropically driven,14,18 though
this is a matter of debate.17 This hypothesis is proposed from
temperature dependence of kcat data from both structural
classes of chorismate mutases. We report herein the thermo-
dynamic parameters, entropic and enthalpic contributions to
the free energy of activation, for both the lyase and mutase
reactions of PchB, and for the uncatalyzed lyase reaction, and
compare these data to those reported previously for the AroQ
and AroH chorismate mutases.
’ MATERIALS AND METHODS
Protein Preparation. Wild-type PchB without a histidine tag was
prepared as previously described.7
Preparation of Isochorismate and Chorismate. Isochoris-
mate was isolated from K. pneumoniae 62-1 harboring the entC
plasmid pKS3-0219 with only minor changes, as described previously.3
Less is known about the structures of the chorismate mutases
from Klebsiella pneumoniae (KpCM; formerly Aerobacter aerogenes)
Received: March 7, 2011
Published: April 19, 2011
r
2011 American Chemical Society
7229
dx.doi.org/10.1021/ja202091a J. Am. Chem. Soc. 2011, 133, 7229–7233
|

Journal of the American Chemical Society
ARTICLE
Scheme 1
where kB is Boltzmann’s constant, h is Planck’s constant, R is the ideal gas
constant, and T is the temperature in Kelvin.
Uncatalyzed Decomposition of Isochorismate in Solution.
Isochorismate undergoes elimination to form salicylate and pyruvate
and rearrangement to form isoprephenate in the absence of enzyme.22
Salicylate formation was monitored in water at temperatures from 30 to
70 °C by monitoring fluorescence with an excitation wavelength of
310 nm and an emission wavelength of 430 nm using a Cary Eclipse
fluorescence spectrophotometer (Varian) and an electrothermal single
cell holder for temperature control ((0.1 °C). The amount of salicylate
formed was determined with a standard curve (0ꢀ50 μM). The rate of
isochorismate disappearance (k) was determined from the plot of
salicylate formation (y) versus time (t) according to the equation:
y ¼ að1 ꢀ eꢀkt
Þ
where a = Io(k1/k), k = k1 þ k2, and Io = 350 μM (the initial
isochorismate concentration). k1 is the rate for the elimination reaction
and k2 is the rate for the Claisen rearrangement. Thermodynamic
activation parameters for the elimination and rearrangement reactions
were calculated from the corresponding Eyring plots.
’ RESULTS AND DISCUSSION
Figure 1
Model for the Entropic Contribution to the Transition
from the EnzymeꢀSubstrate Complex to the Transition
State. The experiments described here and those from previous
work used for comparison all report the temperature dependence
of kuncat or kcat. These data therefore reflect changes in free
energy, enthalpy, and entropy for converting the free substrate
(S) to the free transition state (Sq) in the case of kuncat or
the enzyme substrate complex (ES) to the transition state (ESq) in
the case of kcat. The entropic component for formation of the ESq
for the reactions discussed range from an equal or larger penalty
than that observed for the uncatalyzed reaction to very small, and
in one case, within experimental error of zero. We propose that in
the former instance, where the entropic penalty is large, there
is an enzyme conformational change that is concomitant with
the organization of the substrate (Scheme 1). In this case, the
binding event does not necessarily organize (or fold) the
substrate nor does the loop or lid over the active site close.
Instead, this occurs in subsequent steps which incur an entropic
penalty. In the enzymes for which the entropic contribution to
the ES to ESq transition is small or negligible, the substrate and
enzyme are preorganized by the binding event. In other words,
the binding event folds the substrate and closes the loop or lid
over the active site.
Chorismate (Sigma, 60ꢀ80% pure) was recrystallized as previously
described.20
Chorismate Mutase (CM) Activity Assays. Initial velocities
were measured in 50 mM sodium/potassium phosphate buffer pH 7.5 at
temperatures from 5 to 45 °C by measuring chorismate disappearance at
310 nm with a Cary 50 Bio spectrophotometer (Varian) and an
electrothermal single cell holder for temperature control ((0.1 °C).
Enzyme (80 μM) was incubated for 10 min at the desired temperature,
and the reaction was initiated by the addition of chorismate, varied in
concentration from 0.05 to 1.4 mM. Kinetic data were fit to the
MichaelisꢀMenten equation by the nonlinear regression function of
KaleidaGraph (Synergy Software).
Isochorismate-Pyruvate Lyase (IPL) Activity Assays. Initial
velocities were measured in 50 mM sodium/potassium phosphate buffer
pH 7.5 at temperatures from 5 to 45 °C by measuring the accumulation
of salicylate by fluorescence with an excitation wavelength of 300 nm and
an emission wavelength of 430 nm using a Cary Eclipse fluorescence
spectrophotometer (Varian) and an electrothermal single cell holder for
temperature control ((0.1 °C). Enzyme (0.25 μM) was incubated for
10 min at the desired temperature, and the reaction was initiated by the
addition of chorismate, varied in concentration from 2 to 32 μM. Kinetic
data were fit to the MichaelisꢀMenten equation by the nonlinear
regression function of KaleidaGraph (Synergy Software).
Chorismate Mutase Reaction. In solution, chorismate goes
from a very mobile reactant state (S) to a more ordered transition
state (Sq) for conversion of chorismate to prephenate, and this
change has a ΔSq value of ꢀ12.9 cal/(mol K)23 (Table 1). When
unfolded, the substrate is solvated in a variety of ways, including
structured waters surrounding the hydrophobic expanse. These
structured waters are lost to bulk solvent upon substrate folding
providing a positive change in entropy which is offset wholly or
Calculation of Thermodynamic Activation Parameters.
The activation enthalpy (ΔHq) and entropy (ΔSq) were determined
from the linear regression of the data collected from 5 to 45 °C according
to the Eyring equation:21
ꢀ
ꢁ
kBT
h
ꢀ½ðΔHq=RTÞ ꢀ ðΔSq=RÞꢁ
e
kcat
¼
7230
dx.doi.org/10.1021/ja202091a |J. Am. Chem. Soc. 2011, 133, 7229–7233

Journal of the American Chemical Society
ARTICLE
Table 1. Comparison of the Thermodynamic Parameters for the Mutase and Lyase Reactions
activity
ΔGqa (kcal/mol)
ΔHq (kcal/mol)
ΔSq (cal/(mol K))
structural family
chorismate f prephenate
20.7 þ 0.4
Uncatalyzed rearrangementb
PchB CMc
24.5
19.53 ( 0.01
15.4
17.2
15.0
ꢀ12.9 ( 0.4
ꢀ12.1 ( 0.6
ꢀ9.1 ( 1.2
ꢀ3.0 ( 1.6
ꢀ1.6 ( 1.1
ꢀ1.1 ( 1.2
15.9 ( 0.2
AroQ
AroH
AroQ
AroH
AroQ
B. subtilis CMd
12.7 ( 0.4
16.3 ( 0.5
14.5 ( 0.4
15.9 ( 0.4
E. coli CMe
S. aureofaciens CMf
K. pneumoniae CMf
16.2
chorismate f p-hydroxybenzoate þ pyruvate
23.6 ( 2.8
Uncatalyzed eliminationb
25.7
ꢀ6.9 ( 1.5
isochorismate f isoprephenate
23.48 ( 0.06
Uncatalyzed rearrangementg
25.04 ( 0.03
ꢀ5.22 ( 0.01
isochorismate f salicylate þ pyruvate
21.19 ( 0.03
Uncatalyzed eliminationg
PchB IPLg
25.90 ( 0.02
18.47 ( 0.03
ꢀ15.77 ( 0.02
ꢀ24.3 ( 0.2
11.2 ( 0.1
AroQ
a ΔGq calculated at 298.15 K. bAndrews et al.23 cFigure 2. dKast et al.17 eGalopin et al.18 fGorisch et al.14 gFigure 4.
penalty.14,24 The B. subtilis mutase has ΔSq value which is more similar
to the uncatalyzed reaction than to the other chorismate mutases
(ꢀ9.1 cal/(mol K)),17 leading to the suggestion that the active site
exerts less conformational control and overcomes this deficit with a
more significant change in enthalpy.17 This compensation allows for
the similar ΔGq values for all four mutases (15.0ꢀ17.2 kcal/mol).
The temperature dependence of kcat for the chorismate mutase
activity of PchB (Figure 2), on the other hand, gives a ΔSq value of
ꢀ12.1 cal/(mol K), which is within experimental error of the ΔSq
value of the uncatalyzed reaction. By the same argument, this result
suggests that there is no conformational control of the substrate in the
active site upon formation of the enzymeꢀsubstrate complex. This is
contrary to structural biology data,3,7 which clearly show that the
carboxylates of ligands in the active site are oriented by arginines
(Figure 1C). Nevertheless, the data here indicate that the differing
protein folds (AroQ versus AroH) do not correlate to the differing
conformational control of the substrate in the ES complex. Instead,
these data corroborate the idea that these active sites are indeed
comparable and structural differences do not explain the relative
entropic and enthalpic contributions.
Figure 2. Temperature dependence of kcat for the chorismate mutase
reaction of PchB (O).
Isochorismate-Pyruvate Lyase Reaction. In solution, iso-
chorismate spontaneously undergoes a Claisen rearrangement to
form isoprephenate, which undergoes further reaction to form
3-carboxyphenylpyruvate through the loss of a water molecule
(Figure 3). Isochorismate also spontaneously forms salicylate
and pyruvate in the elimination reaction. Using an NMR assay
and measuring the formation of isoprephenate, 3-carboxyphenylpyr-
uvate, salicylate and pyruvate, Hilvert and colleagues have previously
experimentally determined the ΔGq values for the uncatalyzed reac-
tions at 333.15 K, 24.8 and 26.2 kcal/mol, respectively.22 In the
experiments presented here, the uncatalyzed elimination reaction was
monitored by measuring increasing salicylate fluorescence allowing
for the calculation of the concomitant rearrangement reaction
(Figure 4A), and the thermodynamic parameters were determined
as described in the Materials and Methods (Table 1). The ΔGq
values reported in Table 1 are calculated for 298.15 K for direct
comparison to the previously reported chorismate mutase values.
The ΔGq values for the uncatalyzed reaction calculated for 333.15 K
are in good agreement with the ΔGq values from Hilvert, with the
uncatalyzed rearrangement ΔGq value of 25.2 kcal/mol and the
elimination ΔGq value of 26.4 kcal/mol, but the enthalpic and
entropic contributions are somewhat unexpected. We had expected
partly by negative changes in entropy arising from the restrictions
imposed in folding of the substrate and from solvation of the
developing delocalized charge of the transition state which is not
present in the ground state. Therefore, the observed negative
entropy of activation for the uncatalyzed reaction is a combina-
tion of the effects of such changes as substrate folding, release of
structured waters, and solvation of developing charges.
For the enzymatic conversion of chorismate to prephenate, the
K. pneumoniae and S. aureofaciaens mutases demonstrate a small
change for the conversion from the enzymeꢀsubstrate complex
to the transition state (ꢀ1.1, ꢀ1.6 cal/(mol K), respectively).14 It has
been argued that the observation of little entropic change from ES to
ESq indicates that the chorismate is prearranged in the active site and
only becomes slightly more ordered during this step of the reaction
cycle.14 The E. coli chorismate mutase demonstrates a ΔSq value
of ꢀ3.0 cal/(mol K),18 which is within error of the K. pneumoniae
and S. aureofaciaens mutase values.14 These three enzymes are there-
fore considered to be classic entropy traps: the enzyme achieves
the conformational constriction of the substrate during formation of
the ES so that conversion of ES to ESq incurs no large entropic
7231
dx.doi.org/10.1021/ja202091a |J. Am. Chem. Soc. 2011, 133, 7229–7233

Journal of the American Chemical Society
ARTICLE
Figure 3. Thermal decomposition of isochorismate. Reaction catalyzed
by PchB in bold.
enthalpic and entropic contributions for these reactions to be
comparable to those previously reported for the mutase reaction
(Table 1). However, the entropic penalty for the isochorismate
rearrangement contributes considerably less (ꢀ5.22 cal/(mol K))
than that for the chorismate rearrangement (ꢀ12.9 cal/(mol K),23),
whereas the entropic contribution of the uncatalyzed isochorismate
elimination reaction is the most significant (ꢀ15.77 cal/(mol K)).
Indeed, the entropic penalty for the isochorismate elimination is
most similar to that of the chorismate rearrangement (ꢀ12.9
cal/(mol K)). The thermodynamic parameters of the physiological
lyase activity of PchB are the most surprising (Figure 4B). This
reaction is clearly enthalpically driven, and has a very large entropic
penalty (ꢀ24.3 cal/(mol K)), which is more than 1.5-fold greater
than that of the uncatalyzed reaction (ꢀ15.77 cal/(mol K)).
Rate-Determining Step. Previous work to determine the
rate-determining step for the chorismate mutases comes from
several different protein forms and from many different kinds of
experiments, with the weight of the evidence favoring a chemistry
step that is largely or wholly rate-limiting. First, primary heavy
atom isotope effects documented for the independent EcCM
domain25,26 and BsCM25 were consistent with the hypothesis
that chemistry is significantly rate-limiting. Second, the effects of
viscosogens on steady-state parameters indicate that EcCM is
insensitive to changes in viscosity and thus chemistry is fully rate-
determining, whereas BsCM showed kinetic parameters that
were decreased significantly with increasing glycerol and chem-
istry was calculated to be ∼60ꢀ70% rate-determining.27 The
only caveat is that the uncatalyzed reaction showed a 15%
secondary tritium kinetic isotope effect at C3 and C9, whereas
no comparable effect was detected for the enzyme-catalyzed
reaction of the chorismate mutase-prephenate dehydrogenase
(the bifunctional enzyme of which one domain is a chorismate
mutase with 23% identity and 46% similarity to EcCM).28 If
interpreted in isolation, this would suggest that some physical
step is rate-determining. It would be intriguing to learn if this is
also true of the EcCM domain from the dehydratase enzyme.
The Contribution of Loop or Lid Closing to Catalysis. Hur
and Bruice concluded from molecular dynamics simulations of EcCM
initiated at the loop-closed ES conformer that “...the loop structure at
residues 42ꢀ48 fluctuates more in the [transition state] than in the
[enzymeꢀsubstrate complex],” which led to the argument that the
active site is less stable at the transition state and promotes the idea of a
“near attack conformation” for catalysis.29 In other words, it was
argued that the substrate and enzyme are preorganized by formation
of the ES and formation of the transition state is inconsequential for
catalysis. The theoretical calculations of Skolaski and colleagues
Figure 4. Isochorismate-pyruvate lyase. (A) Temperature dependence of
the uncatalyzed isochorismate elimination reaction ()) and the uncatalyzed
isochorismate rearrangement reaction (4). (B) Temperature dependence
of kcat for the isochorismate-pyruvate lyase reaction of PchB (0).
suggest that Arg90 of BsCM (comparable to the lysine of the active
site loop in the AroQ enzymes) has the greatest stabilization effect and
is responsible (with Arg7) for the preorganization effects of the
substrate.30,31 NMR relaxation dynamics of BsCM showed little
movement at Arg90, which is found in a β-strand.32 However, these
data indicate that the C-terminus serves as a lid and there is
substantial ordering of flexible residues upon ligand binding.32 PchB
shows a dramatic change in the active site between the apo- and
ligand-bound structures with a disorder-to-order transition of the
loop,3,7 promoting the hypothesis that active site loop dynamics are
important for catalysis. These combined data for both the AroH and
AroQ enzymes suggest that loop or lid movement is involved in the
catalytic cycle and should be considered in entropic models
for catalysis. In agreement with this idea, changes to less negative
values of ΔSq have been attributed to decreasing phosphate gripper
loop size in orotidine 50-monophosphate decarboxylases,33 suggest-
ing that active site loop mobility contributes to observed entropy
changes for conversion of the enzymeꢀsubstrate complex to the
transition state.
Conclusions. With the predominance of evidence indicating
that chemistry is rate-limiting and evidence for a conformational
7232
dx.doi.org/10.1021/ja202091a |J. Am. Chem. Soc. 2011, 133, 7229–7233

Journal of the American Chemical Society
ARTICLE
change in the loop or lid closing the active site, we propose
the model shown in Scheme 1. Since the reactant state for kcat is the
enzymeꢀsubstrate complex, a reactive conformation of the sub-
strate may not be formed upon binding of substrate to the enzyme
(9) Lee, A. Y.; Karplus, P. A.; Ganem, B.; Clardy, J. J. Am. Chem. Soc.
1995, 117, 3627–3628.
(10) Chook, Y. M.; Gray, J. V.; Ke, H.; Lipscomb, W. N. J. Mol. Biol.
1994, 240, 476–500.
(11) Chook, Y. M.; Ke, H.; Lipscomb, W. N. Proc. Natl. Acad. Sci.
U.S.A. 1993, 90, 8600–8603.
(Eopen unfolded). Instead, the conformer for catalysis (EclosedSfolded) is
S
formed along the reaction coordinate approaching the transition state
(ESq), or in line with classical transition state theory, the reactive
substrate conformation is formed at the transition state. The ordering
of substrate and loop therefore both contribute to the large entropic
penalty for the overall ES to ESq transition. These substrate and
loop ordering steps may be sequential as shown in the scheme, or
may be concerted: closing of the active site loop may force the
substrate into its folded form for catalysis. For the chorismate mutase
reactions with no entropic penalty for the ES to ESq transition,
substrate binding may promote closing of the active site (forming
(12) Ladner, J. E.; Reddy, P.; Davis, A.; Tordova, M.; Howard, A. J.;
Gilliland, G. L. Acta Crystallogr. 2000, D56, 673–683.
(13) Lee, A. Y.; Stewart, J. D.; Clardy, J.; Ganem, B. Chem. Biol. 1995,
2, 195–203.
(14) Gorisch, H. Biochemistry 1978, 17, 3700–3705.
(15) Camacho, C.; Coulouris, G.; Avagyan, V.; Ma, N.; Papadopoulos,
J.; Bealer, K.; Madden, T. L. BMC Bioinf. 2009, 10, 421.
(16) Huang, X.; Miller, W. Adv. Appl. Math. 1991, 12, 373–381.
(17) Kast, P.; Asif-Ullah, M.; Hilvert, D. Tetrahedron Lett. 1996,
37, 2691–2694.
(18) Galopin, C. C.; Zhang, S.; Wilson, D. B.; Ganem, B. Tetrahedron
Lett. 1996, 37, 8675–8678.
(19) Schmidt, K.; Leistner, E. Biotech. Bioeng. 1995, 45, 285–291.
(20) Rieger, C. E.; Turnbull, J. L. Prep. Biochem. Biotechnol. 1996,
26, 67–76.
Eclosed folded), and therefore, the entropy change for this transition is
S
approximately zero.
’ AUTHOR INFORMATION
(21) Conners, K. A. Chemical Kinetics. The Study of Reaction Rates in
Solution; Wiley-VCH Publishers: New York, 1990.
(22) DeClue, M. S.; Baldridge, K. K.; Kast, P.; Hilvert, D. J. Am.
Chem. Soc. 2006, 128, 2043–2051.
Corresponding Author
lamb@ku.edu
(23) Andrews, P. R.; Smith, G. D.; Young, I. G. Biochemistry 1973,
12, 3492–3498.
’ ACKNOWLEDGMENT
(24) Westheimer, F. H. Adv. Enzymol. Rel. Areas Mol. Biol. 1962,
24, 441–482.
We are grateful to T. C. Gamblin for spectrometer use and
R. L. Schowen for insightful discussions. This publication was
made possible by funds from the Kansas Masonic Cancer
Research Institute, by NIH award number P20 RR016475 from
the INBRE Program of the National Center for Research
Resources, and by NIH award number R01 AI77725 from the
National Institute for Allergy and Infectious Disease.
(25) Gustin, D. J.; Mattei, P.; Kast, P.; Wiest, O.; Lee, L.; Cleland,
W. W.; Hilvert, D. J. Am. Chem. Soc. 1999, 121, 1756–1757.
(26) Wright, S. K.; DeClue, M. S.; Mandal, A.; Lee, L.; Wiest, O.;
Cleland, W. W.; Hilvert, D. J. Am. Chem. Soc. 2005, 127, 12957–12964.
(27) Mattei, P.; Kast, P.; Hilvert, D. Eur. J. Biochem. 1999,
261, 25–32.
(28) Addadi, L.; Jaffe, E. K.; Knowles, J. R. Biochemistry 1983,
22, 4494–4501.
(29) Hur, S.; Bruice, T. C. Proc. Natl. Acad. Sci. U.S.A. 2002,
99, 1176–1181.
(30) Szefczyk, B.; Claeyssens, F.; Mulholland, A. J.; Sokalski, W. A.
Int. J. Quantum Chem. 2007, 107, 2274–2285.
(31) Szefczyk, B.; Mulholland, A. J.; Ranaghan, K. E.; Sokalski, W. A.
J. Am. Chem. Soc. 2004, 126, 16148–16159.
(32) Eletsky, A.; Kienhofer, A.; Hilvert, D.; Pervushin, K. Biochemistry
2005, 44, 6788–6799.
’ ABBREVIATIONS
AroH, chorismate mutase structural family of which BsCM is
a member; AroQ, chorismate mutase structural family of which
EcCM is a member; BsCM, Bacillus subtilis chorismate mutase; CM,
chorismate mutase; EcCM, E. coli chorismate mutase; ES, enzymeꢀ
substrate complex; ESq, transition state of enzyme-catalyzed reaction;
IPL, isochorismate-pyruvate lyase; KpCM, Klebsiella pneumoniae
chorismate mutase; PchB, isochorismate-pyruvate lyase from Pseu-
domonas aeruginosa;Sq, transition state of uncatalyzed reaction;
SaCM, Streptomyces aureofaciens chorismate mutase; TSA, transition
state analogue.
(33) Toth, K.; Amyes, T. L.; Wood, B. M.; Chan, K. K.; Gerlt, J. A.;
Richard, J. P. Biochemistry 2009, 48, 8006–8013.
’ REFERENCES
(1) DeClue, M. S.; Baldridge, K. K.; Kunzler, D. E.; Kast, P.; Hilvert,
D. J. Am. Chem. Soc. 2005, 127, 15002–15003.
(2) Gaille, C.; Kast, P.; Haas, D. J. Biol. Chem. 2002, 277,
21768–21775.
(3) Luo, Q.; Olucha, J.; Lamb, A. L. Biochemistry 2009, 48,
5239–5245.
(4) Gallagher, D. T.; Mayhew, M.; Holden, M. J.; Howard, A.; Kim,
K. J.; Vilker, V. L. Proteins 2001, 44, 304–311.
(5) Nakai, T.; Mizutani, H.; Miyahara, I.; Hirotsu, K.; Takeda, S.;
Jhee, K. H.; Yoshimura, T.; Esaki, N. J. Biochem. 2000, 128, 29–38.
(6) Spraggon, G.; Kim, C.; Nguyen-Huu, X.; Yee, M. C.; Yanofsky,
C.; Mills, S. E. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 6021–6026.
(7) Zaitseva, J.; Lu, J.; Olechoski, K. L.; Lamb, A. L. J. Biol. Chem.
2006, 281, 33441–33449.
(8) Stewart, J.; Wilson, D. B.; Ganem, B. J. Am. Chem. Soc. 1990,
112, 4582–4584.
7233
dx.doi.org/10.1021/ja202091a |J. Am. Chem. Soc. 2011, 133, 7229–7233