Structural analysis of charge discrimination in the binding of inhibitors to human carbonic anhydrases I and II

Article abstract of DOI:10.1021/ja068359w

Despite the similarity in the active site pockets of carbonic anhydrase (CA) isozymes I and II, the binding affinities of benzenesulfonamide inhibitors are invariably higher with CA II as compared to CA I. To explore the structural basis of this molecular recognition phenomenon, we have designed and synthesized simple benzenesulfonamide inhibitors substituted at the para position with positively charged, negatively charged, and neutral functional groups, and we have determined the affinities and X-ray crystal structures of their enzyme complexes. The para-substituents are designed-to bind in the midsection of the 15 A deep active site cleft, where interactions with enzyme residues and solvent molecules are possible. We find that a para-substituted positively charged amino group is more poorly tolerated in the active site of CA I compared with CA II. In contrast, a para-substituted negatively charged carboxylate substituent is tolerated equally well in the active sites of both CA isozymes. Notably, enzyme-inhibitor affinity increases upon neutralization of inhibitor charged groups by amidation or esterification. These results inform the design of short molecular linkers connecting the benzenesulfonamide group and a para-substituted tail group in "two-prong" CA inhibitors: an optimal linker segment will be electronically neutral, yet capable of engaging in at least some hydrogen bond interactions with protein residues and/or solvent. Microcalorimetric data reveal that inhibitor binding to CA I is enthalpically less favorable and entropically more favorable than inhibitor binding to CA II. This contrasting behavior may arise in part from differences in active site desolvation and the conformational entropy of inhibitor binding to each isozyme active site.

Full text of DOI:10.1021/ja068359w

Published on Web 04/04/2007
Structural Analysis of Charge Discrimination in the Binding of
Inhibitors to Human Carbonic Anhydrases I and II
D. K. Srivastava,*,† Kevin M. Jude,§ Abir L. Banerjee,† Manas Haldar,‡
Sumathra Manokaran,† Joel Kooren,† Sanku Mallik,*,‡ and David W. Christianson*,§
Contribution from the Department of Chemistry and Molecular Biology, Department of
Pharmaceutical Sciences, North Dakota State UniVersity, Fargo, North Dakota 58105, and Roy
and Diana Vagelos Laboratories, Department of Chemistry, UniVersity of PennsylVania,
Philadelphia, PennsylVania 19104-6323
Received November 21, 2006; E-mail: dk.srivastava@ndsu.nodak.edu; sanku.mallik@ndsu.nodak.edu; chris@sas.upenn.edu
Abstract: Despite the similarity in the active site pockets of carbonic anhydrase (CA) isozymes I and II,
the binding affinities of benzenesulfonamide inhibitors are invariably higher with CA II as compared to CA
I. To explore the structural basis of this molecular recognition phenomenon, we have designed and
synthesized simple benzenesulfonamide inhibitors substituted at the para position with positively charged,
negatively charged, and neutral functional groups, and we have determined the affinities and X-ray crystal
structures of their enzyme complexes. The para-substituents are designed to bind in the midsection of the
15 Å deep active site cleft, where interactions with enzyme residues and solvent molecules are possible.
We find that a para-substituted positively charged amino group is more poorly tolerated in the active site
of CA I compared with CA II. In contrast, a para-substituted negatively charged carboxylate substituent is
tolerated equally well in the active sites of both CA isozymes. Notably, enzyme-inhibitor affinity increases
upon neutralization of inhibitor charged groups by amidation or esterification. These results inform the
design of short molecular linkers connecting the benzenesulfonamide group and a para-substituted tail
group in “two-prong” CA inhibitors: an optimal linker segment will be electronically neutral, yet capable of
engaging in at least some hydrogen bond interactions with protein residues and/or solvent. Microcalorimetric
data reveal that inhibitor binding to CA I is enthalpically less favorable and entropically more favorable
than inhibitor binding to CA II. This contrasting behavior may arise in part from differences in active site
desolvation and the conformational entropy of inhibitor binding to each isozyme active site.
Introduction
II in the eye lowers intraocular pressure, the primary symptom
of glaucoma. However, since many CA isozymes are expressed
in nearly all tissues where they perform various tissue-specific
functions, the long-term systemic administration of a nonspecific
CA II inhibitor may not only lower intraocular pressure but also
impair the physiological functions of carbon dioxide transport
and/or acid-base balance in other tissues.1a,3 This conundrum
inspired the development of the topically applied CA II
inhibitors dorzolamide and brinzolamide to lower intraocular
pressure in glaucoma patients, since topical administration
minimizes long-term systemic exposure to the inhibitors. Even
so, the systemically administered CA inhibitors acetazolamide,
dichlorophenamide, and methazolamide are approved in the U.S.
for the treatment of epilepsy, glaucoma, high altitude sickness,
and sleep apnea.4 The design of isozyme specific inhibitors
remains a critical challenge in the chemistry and biology of the
carbonic anhydrases.
Due to their involvement in a variety of pathophysiological
processes such as glaucoma, hypertension, convulsion and
epilepsy, altitude sickness, obesity, and diabetes, the carbonic
anhydrases (CAs) have historically served as drug design targets
for the treatment of human diseases.1 However, due to serious
side effects, several highly potent carbonic anhydrase inhibitors
have failed to pass scrutiny at different stages in clinical trials,
and some CA-targeted drugs have been withdrawn from the
market.2 The lack of tissue-selective and isozyme-specific
inhibition of CAs is likely the most prominent reason for
unwanted side effects resulting from systemic administration
of a nonspecific CA inhibitor. For example, inhibition of CA
† Department of Chemistry and Molecular Biology, North Dakota State
University.
‡ Department of Pharmaceutical Sciences, North Dakota State University.
§ University of Pennsylvania.
(1) (a) Schuman, J. Expert Opin. Drug Saf. 2002, 1, 181-194. (b) Gray, W.
D.; Rauh, C. E. J. Pharmacol. Exp. Ther. 1967, 156, 383-396. (c) Barnish,
I. T.; Cross, P. E.; Dickinson, R. P.; Gadsby, B.; Parry, M. J.; Randall, M.
J.; Sinclair, I. W. J. Med. Chem. 1980, 23, 117-121. (d) Pastorekova, S.;
Parkkila, S.; Pastorek, J.; Supuran, C. T. J. Enzyme Inh. Med. Chem. 2004,
19, 199-229. (e) Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. Expert
Opin. Ther. Pat. 2006, 16, 1627-1664.
(2) (a) Herkel, U.; Pfeiffer, N. Curr. Opin. Ophthalmol. 2001, 12, 88-93. (b)
Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. Expert Opin. Ther. Pat.
2004, 14, 667-702.
(3) (a) Maren, T. H. In The Carbonic Anhydrases: New Horizons; Chegwidden,
W. R., Carter, N. D., Edwards, Y. H., Eds.; Birkhauser Verlag: Basel,
2000; pp 425-435. (b) Mansoor, U. F.; Zhang, X.-R.; Blackburn, G. M.
In The Carbonic Anhydrases: New Horizons; Chegwidden, W. R., Carter,
N. D., Edwards, Y. H., Eds.; Birkhauser Verlag: Basel, 2000; pp 437-
459.
(4) Carbonic Anhydrase Inhibitors (Systemic). http://www.mayoclinic.com/
health/drug-information/DR202114 (accessed Nov, 2006).
9
5528
J. AM. CHEM. SOC. 2007, 129, 5528-5537
10.1021/ja068359w CCC: $37.00 © 2007 American Chemical Society

Inhibitor Binding to Carbonic Anhydrases I and II
A R T I C L E S
In the animal kingdom, there are fifteen CA isozymes, of
which five are cytoplasmic (I, II, III, VII, and XIII), two are
mitochondrial (VA and VB), one is secreted (VI), four are
membrane associated (IV, IX, XII, XIV), and three are non-
catalytic (VIII, X, XI).5 Of these isozymes, the X-ray crystal
structures of seven (I, II, III, IV, V, XII, and XIV) have been
determined in the absence and presence of inhibitors.6 Although
these isozymes exhibit varying degrees of amino acid sequence
identity, their active site clefts are remarkably similar and consist
of a catalytic Zn2+ ion situated at the bottom of a 15-Å-deep
conical active site roughly divisible into a hydrophobic half and
a hydrophilic half.6b The Zn2+ ion is coordinated by H94, H96,
H119, and a solvent molecule with a tetrahedral geometry.
The best inhibitors of CAs contain an arylsulfonamide group
that coordinates to the active site Zn2+ ion. General features of
sulfonamide-metal coordination are conserved across all
isozymes of known structure: the ionized sulfonamide NH-
group displaces the zinc-bound hydroxide ion and donates a
hydrogen bond to the side chain of T199, and one sulfonamide
SdO group accepts a hydrogen bond from the backbone NH
group of T199.5,6 The aromatic rings of these inhibitors make
additional weakly polar and van der Waals interactions in the
active site, and ring substituents are capable of van der Waals
and hydrogen bond interactions with residues and solvent
molecules in the midsection of the active site cleft.6
Given that the simplest arylsulfonamide, benzenesulfonamide,
binds to CAs with micromolar affinity, numerous benzene-
sulfonamide derivatives have been synthesized and evaluated
against different carbonic anhydrase isozymes.5 Although many
such inhibitors yield impressive nanomolar binding affinity, they
typically exhibit minimal, if any, specificity for one CA isozyme
versus another. Structure-based approaches7 toward the design
of isozyme-specific inhibitors are potentially facilitated by the
availability of more than 240 crystal structures of CA-inhibitor
complexes in the Protein Data Bank;8 yet attempts to design
isozyme specific inhibitors have met with limited success, e.g.,
usually achieving 10-100-fold differences in binding to one
isozyme relative to another.9 One limitation in the available
structural data is the partial molecular disorder of some,10a-c
but not all,10d of the larger inhibitors observed in crystal
structures. For example, such molecular disorder characterizes
the binding of certain “two-prong” inhibitors containing cupric-
iminodiacetate groups tethered to benzenesulfonamide by 5-12
Å long linker segments.11 This observation now inspires us to
explore the contribution of the length and chemical nature of
the linker segment to enzyme-inhibitor recognition, affinity,
and specificity.
Here, we report the structural and functional consequences
of positively charged, negatively charged, and neutral groups
incorporated at the para position of benzenesulfonamide CA
inhibitors (Table 1). The para-substituted groups are chemically
and electronically diverse and are designed to interact with the
midsection of the active site cleft, i.e., where linker segments
of two-prong benzenesulfonamide inhibitors would bind. Cor-
relation of these structural data with microcalorimetric measure-
ments of inhibitor binding allows us to discern structural and
chemical features of inhibitor linker segments that may con-
tribute to enzyme-inhibitor affinity and specificity.
Results
Binding Affinities of CA Inhibitors. The Ki, Kd, and 1/Ka
values of benzenesulfonamide derivatives determined by kinetic
measurements of p-nitrophenylacetate hydrolysis, the dansyla-
mide displacement assay, and isothermal titration calorimetric
(ITC) studies of CA-inhibitor complexes, respectively, are
recorded in Table 1. The Ki, Kd, and 1/Ka values measured for
each inhibitor are generally comparable, and as a group these
inhibitors exhibit somewhat preferential binding affinity for CA
II than for CA I (a common feature of most benzenesulfonamide
derivatives for which Ki values are reported in the literature5).
However, the degree of isozyme specificity appears to depend
on the para-substituted group for the inhibitors shown in Table
1. For example, the positively charged amino group of pAEBS
(p-aminoethyl benzenesulfonamide) causes ∼6-fold and ∼20-
fold decreases relative to benzenesulfonamide in binding affinity
to CA II and CA I, respectively, indicating that the positively
charged amino group is more poorly tolerated in the CA I active
site than in the CA II active site. In contrast, the negatively
charged carboxylate group of pCEBS (p-carboxyethyl benze-
nesulfonamide) causes slight (∼2-fold) increases in binding
affinity to both isozymes relative to benzenesulfonamide,
indicating that the negatively charged carboxylate is accom-
modated equally well in the CA I and CA II active sites. A
marked increase in the binding affinities of both positively and
negatively charged benzenesulfonamide derivatives results when
(5) (a) Supuran, C. T.; Scozzafava, A.; Conway, J. Carbonic Anhydrase: Its
Inhibitors and ActiVators; CRC Press: Boca Raton, FL, 2004. (b) Supuran,
C. T.; Scozzafava, A.; Casini, A. Med. Res. ReV. 2003, 23, 146-189. (c)
Tripp, B. C.; Smith, K.; Ferry, J. G. J. Biol. Chem. 2001, 276, 48615-
48618.
(6) (a) Kannan, K. K.; Notstrand, B.; Fridborg, K.; Lo¨vgren, S.; Ohlsson, A.;
Petef, M. Proc. Natl. Acad. Sci. U.S.A. 1975, 51-55. (b) Liljas, A.; Kannan,
K. K.; Bergste´n, P.-C.; Waara, I.; Fridborg, K.; Strandberg, B.; Carlbom,
U.; Ja¨rup, L.; Lo¨vgren, S.; Petef, M. Nat. New Biol. 1972, 235, 131-137.
(c) Håkansson, K.; Carlsson, M.; Svensson, L. A.; Liljas, A. J. Mol. Biol.
1992, 227, 1192-1204. (d) Mallis, R. J.; Poland, B. W.; Chatterjee, T. K.;
Fisher, R. A.; Darmawan, S.; Honzatko, R. B.; Thomas, J. A. FEBS Lett.
2000, 237-241. (e) Stams, T.; Chen, Y.; Boriack-Sjodin, P. A.; Hurt, J.
D.; Liao, J.; May, J. A.; Dean, T.; Laipis, P.; Silverman, D. N.; Christianson,
D. W. Protein Sci. 1998, 7, 556-563. (f) Stams, T.; Nair, S. K.; Okuyama,
T.; Waheed, A.; Sly, W. S.; Christianson, D. W. Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 13589-13594. (g) Boriack-Sjodin, P. A.; Heck, R. W.;
Laipis, P. J.; Silverman, D. N.; Christianson, D. W. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 10949-10953. (h) Whittington, D. A.; Waheed, A.;
Ulmasov, B.; Shah, G. N.; Grubb, J. H.; Sly, W. S.; Christianson, D. W.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9545-9550. (i) Whittington, D.
A.; Grubb, J. H.; Waheed, A.; Shah, G. N.; Sly, W. S.; Christianson, D.
W. J. Biol. Chem. 2004, 279, 7223-7228.
(9) (a) Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib. 1999, 14, 343-363.
(b) Popescu, A.; Simion, A.; Scozzafava, A.; Briganti, F.; Supuran, C. T.
J. Enzyme Inhib. 1999, 14, 407-423. (c) Kim, C.-Y.; Whittington, D. A.;
Chang, J. S.; Liao, J.; May, J. A.; Christianson, D. W. J. Med. Chem. 2002,
45, 888-893. (d) Gupta, S. P.; Kumaran, S. J. Enzyme Inhib. Med. Chem.
2005, 20, 251-259. (e) Innocenti, A.; Villar, R.; Martinez-Merino, V.; Gil,
M. J.; Scozzafava, A.; Vullo, D.; Supuran, C. T. Bioorg. Med. Chem. Lett.
2005, 15, 4872-4876. (f) Mincione, F.; Starnotti, M.; Masini, E.;
Bacciottini, L.; Scrivanti, C.; Casini, A.; Vullo, D.; Scozzafava, A.; Supuran,
C. T. Bioorg. Med. Chem. Lett. 2005, 15, 3821-3827. (g) O¨ zensoy, O¨ .;
Puccetti, L.; Fasolis, G.; Arslan, O.; Scozzafava, A.; Supuran, C. T. Bioorg.
Med. Chem. Lett. 2005, 15, 4862-4866. (h) Hillebrecht, A.; Supuran, C.
T.; Klebe, G. ChemMedChem 2006, 839-853.
(10) (a) Boriack, P. A.; Christianson, D. W.; Kingery-Wood, J.; Whitesides, G.
M. J. Med. Chem. 1995, 2286-2291. (b) Bunn, A. M. C.; Alexander, R.
S.; Christianson, D. W. J. Am. Chem. Soc. 1994, 116, 5063-5068. (c)
Elbaum, D.; Nair, S. K.; Patchan, M. W.; Thompson, R. B.; Christianson,
D. W. J. Am. Chem. Soc. 1996, 118, 8381-8387. (d) Alterio, V.; Vitale,
R. M.; Monti, S. M.; Pedone, C.; Scozzafava, A.; Cecchi, A.; De Simone,
G.; Supuran, C. T. J. Am. Chem. Soc. 2006, 128, 8329-8335.
(11) Jude, K. M.; Banerjee, A. L.; Haldar, M. K; Manokaran, S.; Roy, B.; Mallik,
S.; Srivastava, D. K.; Christianson, D. W. J. Am. Chem. Soc. 2006, 128,
3011-3018.
(7) (a) Geysen, H. M.; Schoenen, F.; Wagner, D.; Wagner, R. Nat. ReV. Drug
DiscoV. 2003, 2, 222-230. (b) Jager, S.; Brand, L.; Eggeling, C. Curr.
Pharm. Biotechnol. 2003, 4, 463-476.
(8) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig,
H.; Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235-
242.
9
J. AM. CHEM. SOC. VOL. 129, NO. 17, 2007 5529

A R T I C L E S
Srivastava et al.
Table 1. Binding Affinities of CA Inhibitorsa
a Ki values determined by kinetic assay of p-nitrophenyl acetate hydrolysis; Kd values determined by dansylamide displacement assay; 1/Ka values determined
by isothermal titration calorimetry. b Reference 17b. c Reference 11. d No heat of binding was detected by isothermal titration calorimetry. e Roy, B. C.;
Banerjee, A. L.; Swanson, M.; Jia, X. G.; Haldar, M. K.; Mallik, S.; Srivastava, D. K. J. Am. Chem. Soc. 2004, 126, 13206-13207.
Table 2. Thermodynamic Parameters of CA-Inhibitor
either weaker (benzenesulfonamide) or of insufficient magnitude
to measure (pAEBS and MH 1.25). In the comparison of
inhibitor binding to CA isozymes, then, the most dramatic
differences in ITC binding profiles are noted for pAEBS
and MH 1.25: each of these inhibitors exhibits measurable
heat of binding to CA II, but neither inhibitor exhibits a
measurable heat of binding to CA I. Since pAEBS and MH
1.25 do indeed bind to CA I as demonstrated by kinetic and
dansylamide displacement assays (Table 1), one interpretation
regarding the lack of a significant heat signal could be that the
binding of these inhibitors is dominated by favorable entropic
changes.
Complexation
Ka
(M-
∆
H
°
∆S°
(cal/mol K)
1
enzyme
inhibitor
n
)
(kcal/mol)
CA I
BS
0.9
n.h.a
n.h.a
0.8
0.8
0.8
0.9
0.8
1.0
0.9
(3.8 ( 0.7) × 105 -7.1 ( 0.3
1.7
n.h.a
n.h.a
-1.9
-1.1
-2.1
-5.6
-12
pAEBS
MH 1.25
pCEBS
n.h.a
n.h.a
n.h.a
n.h.a
(1.6 ( 0.3) × 106 -9.0 ( 0.3
(1.1 ( 0.4) × 107 -9.9 ( 0.4
(1.7 ( 0.3) × 106 -9.1 ( 0.3
(2.1 ( 0.1) × 105 -8.9 ( 0.1
(1.0 ( 0.6) × 107 -13 ( 0.6
(2.6 ( 0.4) × 106 -11 ( 0.3
(1.8 ( 0.5) × 107 -11 ( 0.3
MH 1.29
CA II BS
pAEBS
MH 1.25b
pCEBS
-7.7
-3.8
MH 1.29
a No heat of binding was measurable by isothermal titration calorimetry.
b Thermodynamic parameters for the primary binding (a) site.
Figure 1 shows comparative microcalorimetric data for the
binding of benzenesulfonamide to CA I and CA II. The best fit
of the experimental data yields the stoichiometry (n), association
constant (Ka), and enthalpic change (∆H°) of 0.90, 3.8 × 105
M-1, and -7.1 kcal/mol, respectively, against CA I, and
corresponding values of 0.8, 1.7 × 106 M-1, and -9.1 kcal/
mol against CA II. Assuming standard states of 1 M, the above
Ka values yield ∆G° values of -7.6 and -8.5 kcal/mol,
respectively, for binding to CA I and CA II. From these data,
the entropic contributions (∆S°) are deduced to be 1.7 and -2.0
entropy units (cal/mol K), respectively, for inhibitor binding to
CA I and CA II. Although the magnitudes of these values seem
relatively small, they are comparable in magnitude to ∆S values
of 0-7 entropy units estimated for the transfer of a protein-
para-substituted charged groups are neutralized by acylation to
yield MH 1.25 (4-(acetyl-2-aminoethyl)benzenesulfonamide)
and MH 1.29 (p-aminosulfonyldihydrocinnamic acid ethyl ester),
respectively (Table 1).
Analysis of isothermal titration calorimetric data allows us
to dissect enthalpic and entropic contributions to the overall
binding free energy of each enzyme-inhibitor complex that
yields a measurable heat of binding (Table 2). In the case of
CA II, the titration of the enzyme by all benzenesulfonamide
inhibitors yields exothermic peaks. However, in the case of CA
I, except for pCEBS and MH 1.29 the exothermic peaks were
9
5530 J. AM. CHEM. SOC. VOL. 129, NO. 17, 2007

Inhibitor Binding to Carbonic Anhydrases I and II
A R T I C L E S
Figure 2. Isothermal microcalorimetric data for the binding of MH 1.25
to CA II. Note the raw data (top panel) shows two inflection points,
consistent with two-site binding. The solid smooth line is the best fit of the
data according to the two binding site model for the following stoichiom-
etries (n1, n2), association constants (Ka1, Ka2), and enthalpic changes (∆H°1,
∆H°2); 0.8, 1.5, 1.0 × 107 M-1, 2.1 × 105 M-1, -13.0 kcal mol-1, and
-6.1 kcal mol-1, respectively.
whereas binding to CA II is favored by enthalpic contributions
but slightly opposed by entropic contributions.
Thermodynamic parameters are recorded in Table 2 for the
binding of all inhibitors for which heats of binding are
measurable by ITC. With the exception of the CA II-MH 1.25
complex, all binding data conform to the expected 1:1 stoichi-
ometry of each enzyme-inhibitor complex (0.8 < n < 1.0).
As shown in Figure 2, the raw ITC data for the titration of CA
II by MH 1.25 reveal two inflection points consistent with
inhibitor binding to both the active site and a weaker secondary
site near the N-terminus (Vide infra).11 In general, each ITC-
derived 1/Ka value is approximately equal to the corresponding
Ki and Kd value of each inhibitor (Table 1). Irrespective of the
benzenesulfonamide derivative, both ∆H° and ∆S° values are
generally more negative (i.e., enthalpically more favorable and
entropically more disfavored) for binding to CA II compared
with binding to CA I. Thus, the favorable entropy (or the
minimal unfavorable entropy) of inhibitor binding to CA I
(Table 2) is not dependent on the electronic charge of groups
attached to the benzenesulfonamide moiety but instead is
dependent on some feature of the enzyme structure.
Figure 1. Isothermal microcalorimetric titration data for the binding of
benzenesulfonamide (BS) to recombinant human carbonic anhydrase
isozymes I (A) and II (B). The upper and lower panels represent the raw
calorimetric data and fitted data, respectively. The solid smooth lines
represent the best fit of the data for the binding of benzenesulfonamide to
CA I (A) with stoichiometry n ) 0.9, association constant Ka ) 3.8 × 105
M-1, and ∆H° ) -7.1 kcal/mol. The corresponding parameters for the
binding of benzenesulfonamide to CA II are 0.8, 1.7 × 106 M-1, and -9.1
kcal/mol, respectively.
X-ray Crystallographic Studies. High resolution (1.01-1.85
Å) crystal structures were determined for all enzyme-inhibitor
complexes (Tables 3 and 4) except for the CA I-pAEBS
complex, which yielded uninterpretable electron density for the
inhibitor (consistent with low occupancy, a high degree of
molecular disorder, or both). Inhibitor binding does not trigger
any major structural changes in any of the enzyme-inhibitor
bound water molecule to bulk solvent.12 That these values are
opposite in sign indicates that benzenesulfonamide binding to
CA I is favored by both enthalpic and entropic contributions,
(12) Dunitz, J. D. Science 1994, 264, 670.
9
J. AM. CHEM. SOC. VOL. 129, NO. 17, 2007 5531

A R T I C L E S
Srivastava et al.
MH 1.29
Table 3. Crystallographic Data Collection and Refinement Statistics for CA II-Inhibitor Complexes
inhibitor complex
pAEBS
MH 1.25
pCEBS
space group
P21
P21
P21
P21
unit cell constants, (σ) (Å)
a ) 42.289(5)
b ) 41.440(6)
c ) 72.357(9)
â ) 104.558(8)°
1.0000
a ) 42.264(2)
b ) 41.338(4)
c ) 71.975(5)
â ) 104.496(4)°
0.9184
a ) 42.280(2)
b ) 41.361(3)
c ) 72.135(4)
â ) 104.500(3)°
0.9184
a ) 42.339(5)
b ) 41.189(6)
c ) 72.077(8)
â ) 104.838(7)°
1.0000
wavelength (Å)
no. measured reflections
no. unique reflections
max resolution (Å)
245 255
74 655
1.20
0.076 (0.270)
98.8 (95.3)
315 311
105 952
1.03
0.061 (0.283)
89.7 (56.9)
409 170
120 621
1.01
0.064 (0.345)
95.4 (71.1)
216 120
90 979
1.10
0.072 (0.348)
92.7 (87.2)
R
merge (outer shell)a
completeness of data
(outer shell) (%)
no. reflections used in
refinement
73 136
103 824
118 194
89 164
no. reflections in test set
1502
0.1316
0.1647
2375
273
2111
2407
1802
b
Rwork
0.1293
0.1636
2514
0.1214
0.1481
2493
0.1327
0.1637
2429
b
Rfree
no. nonhydrogen atomsc
no. solvent moleculesc
364
340
256
rmsd from ideality
bond lengths (Å)
bond angles (deg)
dihedral angles (deg)
improper dihedral
angles (deg)
0.013
2.3
26.4
1.60
0.014
2.4
26.2
1.68
0.016
2.3
26.0
1.72
0.015
2.4
26.5
1.74
PDB accession codes
2NNG
2NNS
2NNO
2NNV
a Rmerge for replicate reflections, R ) Σ|Ih - Ih |/Σ Ih ; Ih is the intensity measured for reflection h; Ih is the average intensity for reflection h calculated
from replicate data. b Crystallographic R factor, Rwork ) Σ||Fo| - |Fc||/Σ|Fo| for reflections contained in the working set. Free R factor, Rfree ) Σ||Fo| -
|Fc||/Σ|Fo| for reflections contained in the test set held aside during refinement. |Fo| and |Fc| are the observed and calculated structure factor amplitudes,
respectively. c Per asymmetric unit.
Table 4. Crystallographic Data Collection and Refinement
Statistics for CA I-Inhibitor Complexes
of each inhibitor binds identically in the active site: the ionized
sulfonamide NH- group coordinates to Zn2+ and donates a
inhibitor complex
MH 1.25
pCEBS
MH 1.29
hydrogen bond to Oγ of T199, and one sulfonamide oxygen
accepts a hydrogen bond from the backbone NH group of T199.
In CA II-inhibitor complexes, the para-substituted tail of
each inhibitor is well-ordered and associates with the hydro-
phobic side of the active site cleft in the vicinity of P201 and
P202. Polar groups in the tails of three inhibitors (pAEBS,
pCEBS, and MH 1.25) make hydrogen bonds with well-ordered
solvent molecules but make no direct hydrogen bonds with
enzyme residues. The ester moiety of MH 1.29 makes no
hydrogen bond interactions. Each CA II active site also contains
a glycerol molecule from the cryosolvent utilized for flash-
cooling of protein crystals prior to X-ray data collection. This
glycerol solvent molecule presumably displaces water solvent
molecules and forms hydrogen bonds with N67 Nδ2, N67 Oδ1,
Q92 Nꢀ2, and two solvent molecules. The electron density
map of the CA II-MH 1.25 complex (Figure 3a) illustrates
representative features of all four CA II-inhibitor complexes,
and a superposition of all four complexes is found in
Figure 4a.
space group
unit cell constants (Å)
P212121
a ) 62.4(1)
b ) 72.3(1)
P212121
a ) 62.422(8) a ) 62.28(1)
b ) 71.82(1) b ) 71.82(1)
P212121
c ) 122.3(2) c ) 121.96(1) c ) 121.73(1)
1.1271
wavelength (Å)
no. measured reflections 560 061
1.1271
392 855
65 427
1.65
1.0000
181 031
46 613
1.85
no. unique reflections
max resolution (Å)
80 458
1.55
R
merge (outer shell)a
0.097 (0.549) 0.099 (0.451) 0.113 (0.391)
99.9 (99.8)
completeness of data
(outer shell) (%)
no. reflections used in
refinement
97.5 (98.2)
98.6 (96.3)
78 912
61 854
41 995
no. reflections in test set 1581
2516
0.1996
0.2222
4605
1759
0.1947
0.2251
4526
b
Rwork
Rfree
0.2157
0.2477
b
no. nonhydrogen atomsc 4601
no. solvent moleculesc
rmsd from ideality
bond lengths (Å)
bond angles (deg)
dihedral angles (deg)
improper dihedral
angles (deg)
519
525
447
0.005
1.3
24.3
0.77
0.006
1.4
24.2
0.83
0.006
1.3
24.3
0.81
In three of the CA II-inhibitor complexes (MH 1.25, pCEBS,
and MH 1.29), a second inhibitor molecule binds near the
N-terminus of the enzyme at the rim of the active site (data not
shown). The sulfonamide nitrogen forms hydrogen bonds to the
carbonyl oxygen of H15 and to the carboxylate of D19 in a
manner similar to that observed for certain other CA II
inhibitors.11 However, in contrast with previous examples of
inhibitor binding to this “b” site, there are no specific interactions
between the inhibitor tail and the protein. Inhibitor binding to
the weaker “b” site is presumably a consequence of the relatively
PDB accession codes
2NMX
2NN1
2NN7
a Rmerge for replicate reflections, R ) Σ|Ih
-
Ih |/Σ Ih ; Ih is intensity
measured for reflection h; Ih is average intensity for reflection h calculated
from replicate data. b Crystallographic R factor, Rwork ) Σ||Fo| - |Fc||/
Σ|Fo| for reflections contained in the working set. Free R factor, Rfree
)
Σ||Fo| - |Fc||/Σ|Fo| for reflections contained in the test set held aside during
refinement. |Fo| and |Fc| are the observed and calculated structure factor
amplitudes, respectively. c Per asymmetric unit.
complexes studied, and the root-mean-square deviations of
backbone CR atoms between the unliganded enzyme6c,13 and
each enzyme-inhibitor complex range 0.266-0.299 Å for CA
II and 0.307-0.344 Å for CA I. The benzenesulfonamide moiety
(13) Kannan, K. K.; Ramanadham, M.; Jones, T. A. Ann. N.Y. Acad. Sci. 1984,
429, 49-60.
9
5532 J. AM. CHEM. SOC. VOL. 129, NO. 17, 2007

Inhibitor Binding to Carbonic Anhydrases I and II
A R T I C L E S
Figure 3. Omit maps showing the binding of inhibitor MH 1.25 to CA II (a) and CA I (b) contoured at 3σ. The zinc ion appears as a gray sphere and water
molecules appear as red spheres. A glycerol cryosolvent molecule (GOL) also binds in the CA II active site. Hydrogen bond and metal coordination interactions
are designated by red and gray dotted lines, respectively. Figure prepared with Bobscript and Raster3D.27
high concentrations of inhibitors employed in the crystal soaking
protocol used to prepare each crystalline enzyme-inhibitor
complex.
tolerated least well by CA I in comparison with CA II. Second,
inhibitor binding to CA I is generally enthalpically less favorable
and entropically more favorable than inhibitor binding to CA
II. Third, although the benzenesulfonamide rings of different
inhibitors adopt the same general orientation and make the same
intermolecular contacts, their para-substituted tails associate with
opposite walls in the active sites of CA I and CA II. Fourth,
the preferred conformations of para-substituted tails are similar
regardless of whether they are positively charged, negatively
charged, or neutral: inhibitor tails always associate with the
F91-Q92 wall of CA I and the P201-P202 wall of CA II.
All of the inhibitors studied, including the parent unsubstituted
benzenesulfonamide, exhibit slightly higher binding affinities
for CA II than for CA I (Table 1). Isozyme discrimination is
most pronounced for pAEBS, which bears a positively charged
amino substituent, and isozyme discrimination is rather modest
for pCEBS, which bears a negatively charged carboxylate
substituent. Possibly, the sensitivity to the positive charge on
the inhibitor tail results from electrostatic interactions with
histidine residues on the surface of each isozyme. Inhibitor
affinity for each isozyme is enhanced by acylation of pAEBS
and pCEBS to yield neutral inhibitors MH 1.25 and MH 1.29,
respectively. Microcalorimetric data reveal relatively weak
isozyme discrimination for MH 1.25 and slightly stronger
discrimination for MH 1.29, but in neither case does isozyme
discrimination exceed a factor of 10-fold.
The electron density map of the CA I-MH 1.25 complex
(Figure 3b) illustrates representative features observed in all
three CA I-inhibitor complexes. The inhibitors MH 1.25,
pCEBS, and MH 1.29 bind solely in the active site of CA I;
i.e., no binding is observed at a secondary site. The benzene-
sulfonamide moieties of these inhibitors make the same
intermolecular interactions in their CA I complexes as those
observed in their CA II complexes. However, the tails of these
inhibitors adopt significantly different conformations in CA I
compared with CA II: the tails of inhibitors binding to CA I
associate with the F91-Q92 wall of the active site, whereas
the tails of inhibitors bound to CA II associate with the opposite
wall of the active site defined by P201-P202. Other than a
hydrogen bond between the amide carbonyl of MH 1.25 and
the side chain NH2 group of Q92, polar groups in the tails of
these inhibitors do not make any direct hydrogen bond interac-
tions with CA I residues; instead, these substituents typically
make two to three hydrogen bond interactions with well ordered
solvent molecules. The superposition of the three CA I-inhibitor
complexes is found in Figure 4b, where comparison with the
CA II-inhibitor complexes in Figure 4a clearly reveals the
differing conformations of the inhibitor tails in the two isozyme
active sites.
Additional insight on the binding of these inhibitors derives
from the comparison of the enthalpic and entropic changes that
accompany enzyme-inhibitor complexation. The active site
cleft of each CA isozyme is fairly wide (15 Å) and deep (15
Å), roughly divisible into a hydrophobic half and a hydrophilic
half.6 In spite of more negative binding entropies, CA II exhibits
Discussion
Four aspects of structure-affinity relationships are evident
in the binding of differently charged benzenesulfonamide
derivatives to CA I and CA II. First, the free amino group at
the para position of benzenesulfonamide (i.e., pAEBS) is
9
J. AM. CHEM. SOC. VOL. 129, NO. 17, 2007 5533

A R T I C L E S
Srivastava et al.
Figure 4. Stereofigures showing the structures of inhibitor complexes with (a) CA II and (b) CA I. The binding conformations of pAEBS (orange), pCEBS
(purple), and MH 1.29 (green) are superimposed on each isozyme complex with MH 1.25 (white). Note that the para-substituted tails of inhibitors bound
to CA II associate with P201-P202, but the tails of inhibitors bound to CA I associate with F91-Q92 on the opposite wall of the active site. Figure prepared
with Bobscript and Raster3D.27
higher binding affinity than CA I for all of the ligands. This
may derive in part from the presence of a hydrophobic niche in
the midsection of the CA II active site, formed by residues F131,
V135, L198, and L204, that acts as a somewhat constricted
binding site for the p-derivatized inhibitor tails; this niche is
less pronounced in CA I due to the substitutions F131L and
V135A (Figure 4).
The midsection of the CA II active site cleft is slightly more
constricted than that of CA I due in part to the presence of F131
and V135. X-ray crystallographic structure determinations of
unliganded CA I and CA II reveal several ordered water
molecules in each active site.6c,13 The release of these water
molecules to bulk solvent, e.g., as triggered by inhibitor binding,
is generally characterized by an enthalpic cost due to the
disruption of favorable hydrogen bond and van der Waals
interactions, and an entropic gain estimated to be 0-7 entropy
units per solvent molecule released.12,14 The energetics of
active site desolvation are offset by inhibitor binding, which
is expected to yield an enthalpic gain due to new hydrogen
bonds and van der Waals interactions in the complex, and a
net entropic loss due to the fixed position, orientation, and
conformation of the inhibitor in the enzyme active site (despite
a small entropic gain due to additional vibrations in the
enzyme-inhibitor complex).14,15 Except for the binding of
benzenesulfonamide to CA I, the ITC data reported in Table 2
conform to this view of favorable enthalpic and unfavorable
entropic changes. Moreover, the magnitudes of both ∆H° and
∆S° generally appear to be greater for inhibitor binding to CA
II. Indeed, the magnitude of ∆S° appears to be greater for
inhibitor binding to CA II compared with inhibitor binding to
CA I, and especially so for the largest inhibitors MH 1.25 and
MH 1.29. This is consistent with the hypothesis that the
immobilization of inhibitor tails in the F131 hydrophobic niche
contributes to the less favorable entropies of binding to CA II:
the bigger the inhibitor tail, the bigger the entropic cost of
binding.
Krishnamurthy and colleagues describe an additional phe-
nomenon of enthalpy-entropy compensation in the binding of
benzenesulfonamide inhibitors bearing p-substituted oligogly-
cine, oligosarcosine, and oligoethylene glycol chains containing
1-5 residues.14d These investigators find that ∆H° becomes less
favorable and ∆S° becomes more favorable as the length of
the oligomeric tail group increases. In other words, the enzyme-
inhibitor interface is characterized by increasing residual disorder
as the tail group becomes longer, and this is consistent with
the results of X-ray crystallographic experiments.10a,b Strikingly,
∆H° and -T∆S° nearly perfectly compensate each other such
that an essentially invariant ∆G° is measured despite the
different chemical structure of each oligomer and the different
(14) (a) Tanford, C. The Hydrophobic Effect: formation of micelles and
biological membranes, 2nd ed.; Wiley: New York, 1980. (b) Dill, K. A.
Biochemistry 1990, 29, 7133-7155. (c) Rowe, E. S.; Zhang, F.; Leung, T.
W.; Parr, J. S.; Guy, P. T. Biochemistry 1998, 37, 2430-2440. (d)
Krishnamurthy, V. M.; Bohall, B. R.; Semetey, V.; Whitesides, G. M. J.
Am. Chem. Soc. 2006, 128, 5802-5812.
(15) Sturtevant, J. M. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 2236-2240.
9
5534 J. AM. CHEM. SOC. VOL. 129, NO. 17, 2007

Inhibitor Binding to Carbonic Anhydrases I and II
A R T I C L E S
Synthesis of Benzenesulfonamide Derivatives. 4-(Acetyl-2-ami-
noethyl) benzenesulfonamide (MH 1.25) was synthesized by acylation
reaction of the commercially available p-aminoethylbenzenesulfona-
mide. To a solution of 4-(2-aminoethyl) benzenesulfonamide (792 mg,
4 mmol) in DMF (15 mL) acetic anhydride (5 mL) was added, and the
reaction mixture was stirred at room temperature for 14 h. The solvents
were evaporated under reduced pressure. Trace amounts of acetic
anhydride present with the white residue were removed by the addition
of toluene and the evaporation of the solvent under reduced pressure.
The white solid was then dissolved in methanol, and hexane was added
slowly to precipitate the pure product (760 mg, 79%) as a white powder.
1H NMR (400 MHz, CDCl3) δ: 1.77 (s, 3H), 2.75-2.78 (t, 2H), 3.26-
3.29 (q, 2H), 7.28 (s, 2H), 7.38 (d, 2H, J ) 8 Hz), 7.74 (d, 2H, J ) 8
Hz), 7.90-7.94 (m, 1H). 13C NMR (100 MHz, CDCl3) δ: 23.24, 35.51,
40.19, 126.33, 129.73, 142.68, 144.42, 169.73.
number of residues in each oligomer. Krishnamurthy and
colleagues describe this as the “interfacial mobility” model of
binding.14d The implications of these results for the design of
tight-binding enzyme inhibitors are that the conformational
entropy of the linker segment connecting two “prongs” of a
bivalent enzyme inhibitor represents a substantial barrier to the
simultaneous interaction of both prongs. This may explain the
molecular disorder accompanying the binding of some, but not
all, two-prong inhibitors containing different linker segments
to CA II.11 As noted by Krishnamurthy et al., the use of more
rigid linker segments in bivalent enzyme inhibitors would
optimize the enthalpy and entropy of binding.14d
In conclusion, thermodynamic and X-ray crystallographic data
for the binding of differently charged benzenesulfonamide
derivatives to CA I and CA II provide important insight
regarding the design of molecular linkers that can potentially
be used to enhance or alter the affinity and specificity of bivalent
enzyme inhibitors. The results reported herein suggest that
positively charged moieties should be avoided in the design of
linker segments, and such charges are more poorly tolerated
by CA I compared with CA II. The structural basis for such
charge discrimination may be due in part to unfavorable long-
range electrostatic interactions with histidine residues on the
surface of each isozyme. Moreover, the results reported herein
suggest that changes in the conformational entropy of p-
substituted substituents of benzenesulfonamide inhibitors are
more substantial for binding to CA II than to CA I, consistent
with the slightly more constricted midsection of the active site
cleft found in CA II. These insights regarding structure-affinity
relationships for molecular linkers now inform the design of
bivalent benzenesulfonamide inhibitors containing nanoscale
molecular recognition elements, e.g., a fullerene-benzene-
sulfonamide, which we will report in due course.16
The p-carboxyethylbenzenesulfonamide ethyl ester (MH 1.29) was
synthesized from the commercially available free acid p-carboxyeth-
ylbenzenesulfonamide (pCEBS) by the esterification reaction as detailed
below. p-Aminosulfonyldihydrocinnamic acid (250 mg, 1.09 mmol)
and p-toluenesulfonic acid (30 mg, 0.15 mmol) were taken up in ethanol
(25 mL) and heated to reflux for 12 h. After evaporation of the solvent
under reduced pressure, the oily residue was purified by column
chromatography (SiO2/chloroform; Rf ) 0.3). The pure product was
obtained as a white solid (256 mg, 91%). 1H NMR (400 MHz, CDCl3)
δ: 1.18-1.21 (t, 3H), 2.64-2.68 (t, 2H), 2.98-3.01 (t, 2H), 4.06-
4.11 (q, 2H, J ) 7 Hz), 7.39 (d, 2H, J ) 8 Hz), (7.81 d, 2H, J ) 8
Hz). 13C NMR (100 MHz, CDCl3) δ: 17.24, 34.38, 39.01, 64.38,
130.09, 132.78, 145.74, 149.60, 176.98.
Experimental Methods
Materials. Zinc sulfate, ampicillin, chloramphenicol, Tris, and IPTG
were purchased from Life Science Resources, Milwaukee, WI; yeast
extracts and tryptone were purchased from Becton Dickinson, Sparks,
MD; acetonitrile, p-aminoethylbenzenesulfonamide (pAEBS), and
p-carboxyethylbenzenesulfonamide (pCEBS) were from Aldrich Chemi-
cals, Milwaukee, WI; HEPES, p-aminomethylbenzenesulfonamide-
agarose, p-nitrophenyl acetate, and PMSF were obtained from Sigma;
dansylamide was purchased from Avocado Research Chemicals (Hey-
sham, Lancashire, U.K.). All other chemicals were of reagent grade
and were used without further purification.
Methods. The recombinant forms of human CA isozymes I and II
(henceforth simply designated as CA I and CA II) were cloned,
expressed, and purified as described previously.17 The enzyme con-
centrations were determined spectrophotometrically using ꢀ280 ) 49
mM-1 cm-1 and 54 mM-1 cm-1 based on MW ) 30 000 for CA I and
CA II, respectively. The enzyme activities of CA I and CA II were
measured in 25 mM HEPES buffer, pH 7.0, containing 10% acetonitrile
(the standard buffer) at 25 °C, utilizing 0.4 mM p-nitrophenyl acetate
as substrate.18 Unless stated otherwise, all the experiments reported
herein were performed in the above assay buffer.
Spectrophotometric Studies for Determining the Ki Values of
CA-Inhibitor Complexes. The steady-state kinetic experiments for
CA I and CA II catalyzed reactions were performed on a Perkin-Elmer
Lambda 3B spectrophotometer in the standard assay buffer, pH 7.0, as
described previously.17a The initial rates of the enzyme-catalyzed
reactions were measured by following the hydrolysis of the chromogenic
substrate, p-nitrophenyl acetate, at 348 nm. Due to the slow esterase
activity of CA I and CA II, fairly high concentrations of the enzymes
had to be utilized to reliably measure the initial rates of the enzyme
reaction in the absence and presence of benzenesulfonamide derivatives
as inhibitors. Under this situation, we analyzed the inhibition data
(according the competitive inhibition model) by taking into account
(16) Zakharian, T.; Jude, K. M.; Christianson, D. W. Study in progress.
(17) (a) Banerjee, A. L.; Swanson, M.; Roy, B. C.; Jia, X.; Haldar, M. K.; Mallik,
S.; Srivastava, D. K. J. Am. Chem. Soc. 2004, 126, 10875-10883. (b)
Banerjee, A. L.; Eiler, D.; Roy, B. C.; Jia, X.; Haldar, M. K.; Mallik, S.;
Srivastava, D. K. Biochemistry 2005, 44, 3211-3224. (c) Banerjee, A. L.;
Tobwala, S.; Ganguly, B.; Mallik, S.; Srivastava, D. K. Biochemistry 2005,
44, 3673-3682.
(18) Pocker, Y.; Stone, J. T. Biochemistry 1967, 6, 668-678.
9
J. AM. CHEM. SOC. VOL. 129, NO. 17, 2007 5535

A R T I C L E S
Srivastava et al.
the free and bound inhibitor concentrations (eq 1)17a
Wiseman et al.,20 which yields stoichiometry (n), association constant
(Ka), and the standard enthalpy changes (∆H°) for the binding of
inhibitors to CA I and CA II sites. The standard free energy change
(∆G°) for the binding was calculated according to the relationship ∆G°
) -RT ln Ka. Given the magnitudes of ∆G° and ∆H°, the standard
entropy changes (∆S°) for the binding process were calculated according
to the standard thermodynamic equation, ∆G° ) ∆H° - T∆S°.
X-ray Crystallographic Studies of Enzyme-Inhibitor Complexes.
The purified forms of CA I and CA II11,17 were crystallized by the
hanging drop method. For CA II crystallization, 5 µL of protein solution
(10 mg/mL protein, 1 mM methyl mercuric acetate, 50 mM Tris-sulfate,
pH 8.0) and 5 µL of precipitant solution (2.5 M (NH4)2SO4, 50 mM
Tris-sulfate, pH 7.7) were mixed and suspended over a reservoir
containing 1 mL of precipitant solution at 4 °C. Crystals formed within
3 days. Single crystals were transferred to fresh sitting drops containing
precipitant solution plus 1 mM inhibitor (from 20 mM stock in
acetonitrile). After 1 day, crystals were transferred to a drop containing
30% glycerol, 70% precipitant solution, then flash cooled in liquid
nitrogen.
For CA I crystallization, 5 µL of protein solution (11 mg/mL protein,
50 mM Tris-sulfate, pH 8.7) and 5 µL of precipitant solution (23%
polyethylene glycol (PEG) 3350, 200 mM NaCl, and 100 mM HEPES,
pH 7.1) were mixed and suspended over a reservoir containing 1 mL
of precipitant solution at 4 °C. Crystals formed within 2 weeks. Inhibitor
solution (20 mM in acetonitrile) was added to crystallization drops to
a final concentration of 2 mM. Crystals were allowed to equilibrate
for 1 day prior to transfer to 5% glycerol, 95% precipitant solution,
then flash cooled in liquid nitrogen.
V0Ki
V )
K + [I] - 0.5(R - R2 - 4[I] [E] )
x
i
t
t
t
where R ) [I]t + [E]t + Ki
(1)
where V and V0 are initial rates in the presence and absence of inhibitors,
and [I]t, [E]t, and Ki represent total inhibitor, total enzyme, and the
inhibition constant, respectively. The inhibition data were analyzed by
eq 1 using the nonlinear regression analysis software Grafit 4.0 to obtain
the Ki values of inhibitors against both CA I and CA II.
Spectrofluorometric Studies for Determining the Kd values of
CA-Inhibitor Complexes. The spectrofluorometric studies involving
dansylamide as a fluorescence probe were performed on a Perkin-Elmer
lambda 50-B spectrofluorometer in the standard assay buffer pH 7.0.
The binding of dansylamide to carbonic anhydrase results in a blue
shift in the emission spectrum of the fluorophore with a marked
enhancement in its quantum yield.19 Banerjee and co-workers provided
evidence that the latter feature was more pronounced when dansylamide
was bound at the active site of CA I as compared to that of CA II.17c
The dissociation constants of the CA I-dansylamide and CA II-
dansylamide complexes were determined as described previously.17a
The dissociation constants (Kd) of CA-inhibitor complexes were
determined by monitoring the fluorescence signals associated with the
competitive displacement of dansylamide by inhibitors. The data were
fitted by eq 2 using the nonlinear regression analysis software Grafit
4.0
X-ray diffraction data were collected at 100 K at the Advanced Light
Source (Berkeley, CA), beamline 5.0.2, using an ADSC Quantum 315
CCD detector.21 Data were integrated and reduced using HKL200022
or CrystalClear (Rigaku/MSC, The Woodlands, TX). Diffraction data
for each CA II crystal were indexed in space group P21 with one
molecule in the asymmetric unit. Diffraction data for CA I were indexed
in space group P212121 with two molecules in the asymmetric unit.
Unit cell parameters and data reduction statistics are recorded in Tables
3 and 4.
The atomic coordinates of CA II refined at 1.54 Å resolution6c were
obtained from the Research Collaboratory for Structural Bioinformatics
Protein Data Bank (RCSB PDB) (accession code 2CBA)8 and used as
a starting model for crystallographic refinement after deletion of
nonprotein atoms. An initial round of rigid body refinement followed
by simulated annealing and individual B factor refinement was
performed using the program CNS 1.1.23 Model visualization and
rebuilding was performed using the graphics program O 10.24 Locations
of mercury and zinc ions were identified from peaks in |Fo| - |Fc|
maps, and residue 206 was modeled as S-(methylmercury)-cysteine.
Refinement was then continued using the program SHELX-97.25
Anisotropic refinement of all atoms led to a drop in Rfree of 0.0337 for
the CA II-pAEBS complex, 0.0245 for the CA II-MH 1.25 complex,
0.0425 for the CA II-pCEBS complex, and 0.0385 for the CA II-
MH 1.29 complex. The addition of riding hydrogen atoms to each model
resulted in an additional decrease in Rfree of 0.0158 for the CA II-
pAEBS complex, 0.0132 for the CA II-MH 1.25 complex, 0.0132 for
the CA II-pCEBS complex, and 0.0157 for the CA II-MH 1.29
complex. Inhibitor molecules were identified from peaks in |Fo| - |Fc|
maps and were gradually built into the models over several rounds of
â - â2 - 4[E] [B]
x
t
t
[EB] )
(2)
2
where â ) [E]t + [B]t + Kb + (Kb/Kd)[D]t
where E, D, and B are representative of enzyme (CA), dansylamide,
and inhibitor, respectively. The suffix “t” denotes total concentration
of the individual species. Kd and Kb are the dissociation constants of
the enzyme-inhibitor and enzyme-dansylamide complexes, respec-
tively.
Isothermal Titration Calorimetry of Inhibitor Binding to CA
Isozymes. The isothermal titration calorimetric experiments were
performed on an MCS isothermal titration calorimeter (ITC). A
complete description of its predecessor instrument (OMEGA-ITC),
experimental strategies, and data analysis is given by Wiseman et al.20
The calorimeter was calibrated by known heat pulses as described in
the MCS-ITC manual. During titration, the reference cell was filled
with a 0.03% azide solution in water. Prior to the titration experiment,
both the enzyme and ligand solution were thoroughly degassed. The
sample cell was filled either with 1.8 mL (effective volume ) 1.36
mL) of buffer (for control) or with an appropriately diluted enzyme.
The contents of the sample cell were titrated with several aliquots (4
µL each) of the ligand. During the titration, the reaction mixture was
constantly stirred at 400 rpm. The enzyme concentrations were adjusted
by 2% (as recommended by the manufacturer) to include a dilution
effect of the enzyme solution, which occurs following a buffer rinse.
All calorimetric titration data were presented after subtracting the
background signal, deduced from the magnitude of heat pulses at the
end of the titration. The raw experimental data were presented as the
amount of heat produced per second following each injection of ligand
into the enzyme solution (minus the blank) as a function of time. The
amount of heat produced per injection was calculated by integration
of the area under individual peaks by the Origin software. Final data
are presented as the amount of heat produced per injection versus the
molar ratio of ligand to enzyme. The data were analyzed according to
(21) Szebenyi, D. M. E.; Arvai, A.; Ealick, S.; LaIuppa, J. M.; Nielson, C.
Journal of Synchrotron Radiation 1997, 4, 128-135.
(22) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307-326.
(23) Bru¨nger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros, P.;
Grosse-Kunstleve, R. W.; Jiang, J.-S.; Kuszewski, J.; Nilges, M.; Pannu,
N. S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren, G. L. Acta
Crystallogr. 1998, D54, 905-921.
(24) Jones, T. A.; Zou, J.-Y.; Cowan, S. W.; Kjeldgaard, M. Acta Crystallogr.
1991, A47, 110-119.
(25) Sheldrick, G. M.; Schneider, T. R. Methods Enzymol. 1997, 277, 319-
343.
(19) Chen, R. F.; Kernohan, J. C. J. Biol. Chem. 1967, 242, 5813-5823.
(20) Wiseman, T.; Williston, S.; Brandts, J. F.; Lin, L. N. Anal. Biochem. 1989,
179, 131-137.
9
5536 J. AM. CHEM. SOC. VOL. 129, NO. 17, 2007

Inhibitor Binding to Carbonic Anhydrases I and II
A R T I C L E S
refinement; restraints on inhibitor bond angles and distances were taken
from similar structures in the Cambridge Structural Database,26 and
standard restraints were used on protein bond angles and distances
throughout refinement. Alternate conformations of disordered side
chains and inhibitor molecules were modeled by fitting to positive and
negative peaks in |Fo| - |Fc| maps. Water molecules were built into
peaks >3σ in |Fo| - |Fc| maps that demonstrated appropriate hydrogen-
bonding geometry.
The atomic coordinates of orthorhombic CA I refined at 1.55 Å
resolution (PDB accession code 2FOY)11 were used as a starting model
for crystallographic refinement after deleting nonprotein atoms. An
initial round of rigid body refinement followed by simulated annealing
and individual B factor refinement was performed using the program
CNS 1.1.23 Model visualization and rebuilding were performed using
the graphics program O.24 Locations of zinc ions were identified from
peaks in |Fo| - |Fc| maps. Water molecules that demonstrated
appropriate hydrogen-bonding geometry were built into peaks >3σ in
|Fo| - |Fc| maps. Inhibitor molecules were identified from peaks in
|Fo| - |Fc| maps; refinement restraints on inhibitor bond angles and
lengths were taken from the refined models of the CA II complexes.
Final refinement statistics for all structures of CA I- and CA II-
inhibitor complexes are presented in Tables 3 and 4. The atomic
coordinates of each structure have been deposited in the PDB with the
accession codes listed in Tables 3 and 4.
Acknowledgment. This research was supported by the
National Institutes of Health Grants CA 113746 to D.K.S. and
S.M., GM 63404 to S.M., and GM 49758 to D.W.C. D.W.C.
was also supported in part by the Underwood Fellowship from
the Biotechnology and Biological Sciences Research Council
for sabbatical study in the Department of Biochemistry,
University of Cambridge, where he also thanks Professor Sir
Tom Blundell for stimulating scientific discussions. The crystal-
lographic data were collected at the Cornell High Energy
Synchrotron Source (CHESS), which is supported by the
National Science Foundation Grant DMR-0225180 and the
National Institute of Health Grant RR-01646.
(26) Allen, F. H. Acta Crystallogr. 2002, B58, 380-388.
(27) (a) Merritt, E. A.; Murphy, M. E. P. Acta Crystallogr. 1994, D50, 869-
873. (b) Kraulis, P. J. J. Appl. Crystallogr. 1991, 24, 946-950. (c) Esnouf,
R. M. J. Mol. Graphics Modell. 1997, 15, 132-134.
JA068359W
9
J. AM. CHEM. SOC. VOL. 129, NO. 17, 2007 5537