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Contributions of Long-Range Electrostatic Interactions to 4-Chlorobenzoyl-CoA
Contributions of Long-Range Electrostatic Interactions to 4-Chlorobenzoyl-CoA
Dehalogenase Catalysis: A Combined Theoretical and Experimental Study Jingbo Wu, Dingguo Xu, Xuefeng Lu, Canhui Wang, Hua Guo,* and Debra Dunaway-Mariano*
Department of Chemistry, Uni</i>V<i>ersity of New Mexico, Albuquerque, New Mexico 87131
Recei</i>V<i>ed July 26, 2005; Re</i>V<i>ised Manuscript Recei</i>V<i>ed October 17, 2005
ABSTRACT
: It is well established that electrostatic interactions play a vital role in enzyme catalysis. In this
work, we report theory-guided mutation experiments that identified strong electrostatic contributions of
a remote residue, namely, Glu232 located on the adjacent subunit, to 4-chlorobenzoyl-CoA dehalogenase
catalysis. The Glu232Asp mutant was found to bind the substrate analogue 4-methylbenzoyl-CoA more
tightly than does the wild-type dehalogenase. In contrast, the k
cat
for 4-chlorobenzoyl-CoA conversion to
product was reduced 10000-fold in the mutant. UV difference spectra measured for the respective enzyme-
ligand complexes revealed an
3-fold shift in the equilibrium of the two active site conformers away
from that inducing strong
-electron polarization in the ligand benzoyl ring. Increased substrate binding,
decreased ring polarization, and decreased catalytic efficiency indicated that the repositioning of the point
charge in the Glu232Asp mutant might affect the orientation of the Asp145 carboxylate with respect to
the substrate aromatic ring. The time course for formation and reaction of the arylated enzyme intermediate
during a single turnover was measured for wild-type and Glu232Asp mutant dehalogenases. The
accumulation of arylated enzyme in the wild-type dehalogenase was not observed in the mutant. This
indicates that the reduced turnover rate in the mutant is the result of a slow arylation of Asp145, owing
to decreased efficiency in substrate nucleophilic attack by Asp145. To rationalize the experimental
observations, a theoretical model is proposed, which computes the potential of mean force for the
nucleophilic aromatic substitution step using a hybrid quantum mechanical/molecular mechanical method.
To this end, the removal or reorientation of the side chain charge of residue 232, modeled respectively by
the Glu232Gln and Glu232Asp mutants, is shown to increase the rate-limiting energy barrier. The calculated
23.1 kcal/mol free energy barrier for formation of the Meisenheimer intermediate in the Glu232Asp mutant
represents an increase of 6 kcal/mol relative to that of the wild-type enzyme, consistent with the 5.6
kcal/mol increase calculated from the difference in experimentally determined rate constants. On the basis
of the combination of the experimental and theoretical evidence, we hypothesize that the Glu232(B) residue
contributes to catalysis by providing an electrostatic force that acts on the Asp145 nucleophile.
The enzyme 4-chlorobenzoyl-CoA (4-CBA-CoA)
1
deha-
logenase is found in 4-chlorobenzoate-degrading bacteria (1,
2) wherein it catalyzes the hydrolytic dehalogenation of
4-chorobenzoyl-CoA (4-CBA-CoA). Because of the unique-
ness of the nucleophilic aromatic substitution reaction
catalyzed and the potential application of this enzyme in the
bioremediation of halogenated aromatics (3, 4), much effort
has been devoted to understanding its catalytic mechanism
(5-22). The dehalogenation reaction proceeds via two partial
reactions: nucleophilic aromatic substitution (S
N
Ar) (23),
followed by ester hydrolysis (Scheme 1). In the first partial
reaction an active site residue (Asp145) attacks the C(4)
position of the substrate benzoyl ring in the enzyme-
substrate complex (ES) to form the enzyme-Meisenheimer
complex (EMc). The expulsion of the chloride ion yields
the arylated enzyme complex (EAr). This intermediate is then
hydrolyzed by a His90-bound water molecule forming
4-hydroxylbenzoyl-CoA (4-HBA-CoA), which is subse-
quently released from the enzyme.
The high-resolution X-ray structure of wild-type dehalo-
genase complexed with 4-HBA-CoA (EP) has shown that
the active site is formed at the subunit interface of the
homotrimer (Figure 1A) (24). The substrate adopts a U-
shaped conformation with the benzoyl moiety extending into
a deep active site cavity, in close proximity of the Asp145
nucleophile. The benzoyl carbonyl oxygen is engaged in
hydrogen bond interactions with the backbone amide NHs
of Gly114 and Phe64 (Figure 1B) and electrostatic interaction
with the positive pole of the active site R-helix (pictured in
Figure 1A), which terminates in Gly114. Collectively, these
interactions comprise what will be referred to hereafter as
the oxyanion hole. The oxyanion hole effect is amplified
by the network of hydrogen bonds that extend from this site This work was supported by NIH Grant GM28688 to D.D.-M. and
by NSF Grant MCB-0313743 to H.G.
* To whom correspondence should be addressed. D.D.-M.: tel, 505-
277-3383; fax, 505-277-6202; e-mail, dd39@unm.edu. H.G.: tel, 505-
277-1716; fax, 505-277-2609; e-mail, hguo@unm.edu.
1
Abbreviations: 4-CBA, 4-chlorobenzoate; 4-CBA-CoA, 4-chloro-
benzoyl-coenzyme A; 4-HBA, 4-hydroxybenzoate; 4-HBA-CoA, 4-hy-
droxybenzoyl-coenzyme A; 4-MBA-CoA, 4-methylbenzoyl-coenzyme
A; ES, enzyme-substrate complex; EP, enzyme-product complex;
EMc, enzyme-Meisenheimer complex; EAr, arylated enzyme complex;
DTT, dithiothreitol; K
+
Hepes, potassium salt of N-(2-hydroxyethyl)-
piperazine-N -2-ethanesulfonate; PMF, potential of mean force; GSD,
ground state destabilization; NAC, near-attack conformation.
102
Biochemistry 2006, 45, 102-112
10.1021/bi051477w CCC: $33.50
© 2006 American Chemical Society
Published on Web 12/06/2005 (22, 24, 25). The aromatic side chains that closely encircle
the benzoyl ring (Figure 1B) might serve to enhance the
-electron pull effect of the oxyanion hole at one end of the
benzoyl ring and the
-electron push effect of the Asp145
carboxyl anion directed at the opposite end of the ring (11).
The degree of polarization of the benzoyl
-electrons, as
Scheme 1: Mechanism of 4-CBA-CoA Dehalogenase Catalysis
F
IGURE
1: (A) Ribbon representation of the 4-CBA-CoA dehalogenase trimer generated from the X-ray coordinates of the wild-type 4-CBA-
CoA dehalogenase-4-HBA-CoA complex (24). Chains A, B, and C are shown in green, orange, and cyan, respectively. 4-HBA-CoA is in
black, the Glu232 R-helix is in magenta, and the Gly114 R-helix is in lime. (B) Active site of the 4-CBA-CoA dehalogenase-4-HBA-CoA
complex (24). The ligand 4-HBA-CoA is shown in black. The side chains of the aromatic residues Phe64 (orange), Phe82 (green), Trp89
(green), and Trp137 (green) form the hydrophobic sheath. The Asp145 nucleophile is colored orange, the His90 base yellow, the H-bond
donor Gly114 orange, the Thr146 green, and the R-helix green. Oxygen atoms are in red, and nitrogen atoms are in blue. Dashed lines
signify hydrogen bonds. (C) Representation of three conformations of the Asp145 side chain. The side chain of Asp145 observed in the
X-ray structure of the wild-type dehalogenase-4-HBA-CoA complex (24) is colored magenta. The side chain of Asp145 observed in the
X-ray structure of the W137F dehalogenase-4-HBA-CoA and W137F dehalogenase-4-MBA-CoA complexes is colored lime (H. M.
Holden and M. M. Benning, unpublished results). For the NAC conformation, the Asp145 side chain is colored cyan. To generate this
conformation, the side chain of Asp145 was manually rotated from its position in the 4-HBA-CoA complex [viz. as in (B)] (24) to satisfy
the conditions of the NAC conformation (26, 32). The side chains of His90, Trp137, and Glu232(B), as observed in the X-ray crystal
structure of the 4-CBA dehalogenase-4-HBA-CoA complex (24), are colored yellow, and the backbone of Thr146 is colored cyan. Dashed
lines signify hydrogen bonds.
Electrostatic Interactions in Dehalogenase Catalysis
Biochemistry, Vol. 45, No. 1, 2006 103 measured by Raman difference spectroscopy (16), has been
correlated with the efficiency of EMc formation. The
oxyanion hole binds the EMc more tightly than it does the
substrate (19). In contrast, the Asp145 point charge appears
to destabilize the ES complex (16). Specifically, replacement
of Asp145 with a neutral amino acid increases the substrate
binding affinity while it decreases the polarization of the
benzoyl ring (17). Because the charge on the Asp145 residue
dissipates into the entire benzoyl moiety as the transition
state is reached, the electrostatic interaction might contribute
to a reduction in the energy barrier.
Theoretical studies have indicated that the Asp145 side
chain assumes a near attack conformation (NAC) prior to
its attack at C(4) of the substrate ring (26, 27). This
conformation, which is stabilized by hydrogen bond interac-
tion with the backbone amide of Thr146, differs from that
observed in the crystal structure of the wild-type dehaloge-
nase-4-HBA-CoA complex, which is stabilized by hydrogen
bond in