A simple carbonic anhydrase model which achieves catalytic hydrolysis by the formation of an 'enzyme-substrate'-like complex

Article abstract of DOI:10.1039/b107683n

One of the attractive features of biomimetic catalysts is their amenability, relative to their enzyme counterparts, to the testing of structural and mechanistic hypotheses. Thus, the reproduction of the active site of an enzyme in a model system provides

Full text of DOI:10.1039/b107683n

View Article Online / Journal Homepage / Table of Contents for this issue
A simple carbonic anhydrase model which achieves catalytic
hydrolysis by the formation of an ‘enzyme–substrate’-like
complex†
2
Timothy G. Sprigings*‡ and C. Dennis Hall
King’s College, University of London, Department of Chemistry, Strand, London,
UK WC2R 2LS
Received (in Cambridge, UK) 23rd August 2001, Accepted 10th September 2001
First published as an Advance Article on the web 16th October 2001
One of the attractive features of biomimetic catalysts is their amenability, relative to their enzyme counterparts, to the
testing of structural and mechanistic hypotheses. Thus, the reproduction of the active site of an enzyme in a model
system provides a tool which is free of much of the complexity of the enzyme. Using this approach, we describe a
new model of the active site of the hydrolytic enzyme carbonic anhydrase (CA). The model (1) is composed of a Zn
complex of the tripodal ligand 1,1,1-tris(aminomethyl)ethane. We find that, in analogy to CA, the complex possesses
2ϩ
2ϩ
a water molecule whose pK is reduced to 8.0 by coordination to the chelated Zn ion. We demonstrate that the
a
complex catalyses the hydrolysis of a model ester substrate (p-nitrophenyl acetate, p-NPA) with a second-order rate
Ϫ1 Ϫ1
constant (k ) of 0.71 M
s
(55.0 ЊC, pH 8.20, ionic strength, I = 0.1 M, aqueous solution), and moreover that it
2
does so with Michaelis–Menten kinetic behaviour (K = 7.6 mM; 45.0 ЊC, pH 8.20, I = 0.1 M, 50% v/v CH CN–H O).
m
3
2
The comparison of these data with those for CA suggests that the hydrophobic cavity and Thr199 residue (which lie
2
ϩ
adjacent to the active-site of the enzyme) contribute only marginally to the pK reduction of the Zn -bound water
a
2ϩ
molecule. Despite the absence of these moieties, the chelated Zn ion is still capable of forming an ‘enzyme–
substrate’-like complex, but the stability of the complex is approximately one order of magnitude smaller than
that of the enzyme.
Introduction
1
Carbonic anhydrase (CA) is a prominent member of the
2ϩ
family of Zn hydrolase enzymes, which are widely-employed
in nature to catalyse the hydrolysis and hydration of a
2
variety of substrates. It has the highest turnover number of all
6
Ϫ1
known enzymes (1.4 × 10 s at 25 ЊC for human CA isozyme
1a
II), and as such approaches the limits of diffusion control and
enzymatic perfection. The key to the hydrolytic activity of the
2ϩ
enzyme is a Zn ion, which is chelated in a tetrahedral array
by three histidine residues and a water molecule (Fig. 1). The
2ϩ
Lewis acidity of the chelated Zn ion serves to lower the pKa
of the bound water to ca. 7.5, furnishing an enhanced con-
1,3
centration of hydroxide ions at the physiological pH. It
is widely-believed that carbonic anhydrase carries out the
efficient hydration of carbon dioxide to hydrogen carbonate by
2ϩ
the nucleophilic attack of the Zn -bound hydroxide ion on the
1
substrate.
The mechanism is outlined in greater detail in Scheme 1. The
coordinated hydroxide ion is stabilised by hydrogen-bonding
with a neighbouring threonine residue (Thr199), which is
itself activated by Glu106. The whole array is encased within a
Fig. 1
Ϫ
Michaelis–Menten kinetic behaviour). The HCO3 pro-
duced diffuses from the pocket, and is replaced by a molecule of
water. The Zn ion binds a further molecule of water, which is
1
7 Å-deep cavity, the walls of which are lined with hydrophobic
residues, which are particularly prevalent in the region of the
deep’ water. The substrate displaces the ‘deep’ water, thus
2ϩ
deprotonated in the final, rate-limiting, step of the catalytic
‘
2ϩ
2ϩ
cycle, the lability of the Zn ion facilitating the Zn –H O ↔
2
positioning itself closely to the zinc-bound hydroxide ion. The
hydration reaction is facilitated by the proximate orientation of
2ϩ
Ϫ
2ϩ
4
Zn –OH ↔ Zn interconversion. The enzyme is clearly
tailored for the catalysis of the CO –HCO3 interconversion,
but nonetheless it demonstrates considerable promiscuity in
the accommodation of other substrates. Thus, CA catalyses
the hydrolysis of carboxylic§ and carbonate esters, acid
Ϫ
2
2ϩ
the Zn -bound hydroxide ion to the enzyme–substrate com-
plex, which is very short-lived and therefore cannot be observed
directly (although its presence is inferred by the observation of
†
Electronic supplementary information (ESI) available: experimental
data. See http://www.rsc.org/suppdata/p2/b1/b107683n/
Present address: Institute of Chemistry and Cell Biology, Harvard
Medical School, 250 Longwood Avenue, SGM 604, Boston, MA,
2115-5731, USA.
§ Including p-nitrophenyl acetate, which is widely-used in kinetic
studies of CA because the p-nitrophenolate anion produced at pH > 7 is
strongly chromophoric (λmax ≈ 405 nm, ε = 2.1 × 10 M cm ), and the
rate of reaction is such that the use of stopped-flow techniques is not
necessary.
4
Ϫ1
Ϫ1
‡
0
DOI: 10.1039/b107683n
J. Chem. Soc., Perkin Trans. 2, 2001, 2063–2067
This journal is © The Royal Society of Chemistry 2001
2063

View Article Online
Fig. 2 Plots obtained for the titration of (A) tris(aminomethyl)ethaneؒ
3
HClO (1 mM) and (B) tris(aminomethyl)ethaneؒ3HClO (1 mM) ϩ
4 4
2ϩ
Zn (1 mM) against NaOH (0.1 M). (25 ± 0.1 ЊC, aqueous, I = 0.1 M).
Results and discussion
Scheme 1 Proposed mechanism for the hydration of CO by carbonic
2
The characterisation of complex 1
anhydrase.
1
H NMR. A solution of complex 1 (1 : 1 tris(aminomethyl)-
2ϩ
chlorides, sulfonate esters, sulfonyl chlorides and dialkyl
monoaryl phosphate esters, and the hydration of aldehydes and
ethane–Zn ) suitable for NMR analysis was prepared as
1
described in the Experimental section. The H NMR spectrum
(recorded at pH 8.8 in 20% v/v D O–CD CN) indicates the
5
pyruvic acid.
2
3
In order to explore the active-site ‘core’ required for the activ-
presence of predominantly (>95%) one species. Three pro-
ity of CA, several excellent model systems have been described
nounced signals are observed, viz. the apical methyl protons at
2ϩ
(
Fig. 1). In addition to structural models, which demonstrate
0.57 ppm (0.83 ppm in the absence of Zn ), the methylene
2ϩ
2ϩ
the facility with which Zn forms tetrahedral complexes com-
posed of three nitrogen atoms and an apical water molecule or
protons at 2.97 ppm (2.58 ppm in the absence of Zn ) and the
amino protons at 2.77 ppm (not observed in the absence of
6
2ϩ
hydroxide ion, the hydrolytically-active model (2) reported by
Zn ). The simplicity of the spectrum indicates that a single,
Ruf and Vahrenkamp cleaves activated esters in hydrophobic
solvents such as benzene and chloroform, albeit in a stoichio-
metric reaction. Other model complexes have been shown
strong complex is formed. The alternative explanation (time-
averaged signals due to multiple complexes which interchange
rapidly on the NMR timescale) is discounted by the observ-
7
to possess CA-like activity in an aqueous or semi-aqueous
ation of distinct NH and HOD signals, since the rate of proton
2
8
environment. Perhaps the example most faithful to CA is that
exchange between NH and HOD would be expected to be
2
2
ϩ
described by Kimura et al., which consists of a Zn complex
3) of the macrocyclic polyamine (‘azacrown’) 1,5,9-triaza-
greater than the rate of complex interconversion.
(
9
2ϩ
cyclododecane. The complex was shown to catalyse the hydra-
tion of acetaldehyde and the hydrolysis of methyl acetate, the
pH–rate profile of the latter reaction being sigmoidal in
shape, with a point of inflection at pH 7.3, corresponding to the
Potentiometric titration. Having established that Zn and
tris(aminomethyl)ethane form essentially one stable complex,
potentiometric titrations were performed to explore the nature
2ϩ
of the Zn -binding moieties. Plots (Fig. 2) were obtained for
2ϩ
pK of the Zn -bound hydroxide ion. Another complex, 4,
the titration of a solution of tris(aminomethyl)ethaneؒ3HClO4
a
reported recently by Bazzicalupi et al. possesses a poly-
(A), and a solution of tris(aminomethyl)ethaneؒ3HClO con-
4
ether chain which partially models the hydrophobicity of the
taining one equivalent of Zn(ClO ) (B), against 0.1 M sodium
hydroxide (expressed as equivalents of OH , a). Plot (A) clearly
shows three points of inflection corresponding to the sequential
4
2
10
Ϫ
enzyme active site. The model catalyses the hydrolysis of p-
nitrophenyl acetate (p-NPA) with a sigmoidal pH–rate profile
2ϩ
whose point of inflection corresponds to the pK of the Zn -
deprotonation of the nitrogen atoms of the ligand. The pK
a
a
1
2
bound hydroxide ion (8.8) which was suggested by potentio-
metric titration.
values for tris(aminomethyl)ethane (pK = 10.4, pKa = 8.6,
a
3
pKa = 5.8) which are indicated by the plot are in agreement
with literature values. Plot (B) indicates that an additional
equivalent of OH is consumed in the presence of Zn . Of the
11
13
These, and other examples illustrate that simple model sys-
tems are capable of mimicking the behaviour of large, complex
enzymes remarkably well. The results reported so far support
the proposed mechanism of CA, but Michaelis–Menten kinetic
Ϫ
2ϩ
four pK values, the lowest two correspond almost exactly to
a
those of (A), and are attributed to the deprotonation of the two
12
behaviour (a key aspect of enzyme-catalysed reactions) has
not been reproduced in a model system. Consequently, the
extent to which the hydrophobic cavity and Thr199 contribute
to the formation of the ‘enzyme–substrate’ complex has been
least basic nitrogen atoms. The pK of the most basic nitrogen
a
atom is reduced from 10.4 to 7.3, reflecting the complete
2ϩ
chelation of the Zn ion by the three donors. The fourth
Ϫ
equivalent of OH corresponds to pK = 8.0, and is attributed
a
2
ϩ
2ϩ
less well understood. Herein, we report that the Zn complex
1) of the tripodal ligand 1,1,1-tris(aminomethyl)ethane is a
to the deprotonation of a Zn -bound water molecule, suggest-
(
ing that complex 1 could function as a CA-like hydrolytic
good model of CA. The complex catalyses the hydrolysis of the
ester p-NPA at approximately physiological pH and does so
with Michaelis–Menten kinetic behaviour, the magnitude of
which suggests that a relatively ‘naked’, chelated Zn ion is
itself capable of forming an ‘enzyme–substrate’-like complex,
albeit with a stability which is about 13 times less than that of
the enzyme.
catalyst with a rate optimum above pH ≈ 8.
The apparent pKa (8.0) is not as close to that of the
enzyme (7.5) as is that in the case of 3 (7.3), but is closer than
2ϩ
2ϩ
14
the corresponding values for Zn(H O)6 (9.6) and 4 (8.8).
2
The amine donors of tris(aminomethyl)ethane are not held in
the relatively rigid macrocyclic arrangement of those belonging
to the ligand in 3, but they are free to adopt a similar tripodal
2
064
J. Chem. Soc., Perkin Trans. 2, 2001, 2063–2067

View Article Online
Fig. 4 Michaelis-Menten plot for the liberation of p-nitrophenolate
Fig. 3 pH–rate profile for the liberation of p-nitrophenolate from
p-NPA in the presence of 1 (1 mM) ϩ buffer (50 mM) (᭹), and in the
presence of buffer only (50 mM) (᭿). ([p-NPA] = 20 µM, 55 ± 0.1 ЊC,
aqueous, buffer = MES, MOPS or CHES, I = 0.1 M.)
from p-NPA by 1 (50 µM). (pH 8.20, 45 ± 0.1 ЊC, 50% v/v CH CN–
3
H O, 50 mM MOPS buffer, I = 0.1 M.) ∆A.U. = ∆Absorbance (arbitrary
2
units).
stability of the ‘enzyme–substrate’ complex, leading to a reduc-
configuration, and this is particularly likely in the presence of a
species (such as a proton or Zn ion) to which they can chelate.
2ϩ
tion in the value of K . This finding is in agreement with a
m
study in which site-directed mutagenesis was used to alter the
This is manifested by the distribution of the pK values of
a
18
nature of the hydrophobic cavity of the enzyme itself.
tris(aminomethyl)ethane over a wide pH range, in common
with macrocyclic polyamines (such as the ligand alone in 3), in
which the amine donors ‘cooperate’ to bind a single proton. In
The most plausible substrate-binding moiety for the form-
ation of the ‘enzyme–substrate’-like complex with 1 is the
2ϩ
2ϩ
Zn ion itself. The binding of the carbonyl oxygen atom of the
2ϩ
contrast, the relatively high pK of the Zn -bound water mol-
a
substrate to the Zn ion would position the carbonyl carbon
atom of the substrate immediately adjacent to the nucleophilic
hydroxide ion. The attack of the hydroxide ion on the substrate
would thus be highly-favoured, since this would approximate to
an intramolecular reaction. The formation of a five-coordinate
‘enzyme–substrate’-like complex is consistent with the well-
ecule in 4 may arise from the non-tripodal nitrogen donor array
of the parent ligand. We conclude that the hydrophobic cavity
2ϩ
and Thr199 of CA do not greatly influence the pK of the Zn -
a
bound water molecule, and that this is more significantly
affected by the relative geometries of the three donor nitrogen
14–16
atoms.
2ϩ
established amenability of Zn to changes in coordination
14,19
number.
The demonstration of an ‘enzyme–substrate’-like
Complex 1 as a catalyst for ester hydrolysis
complex using the simple model 1 therefore suggests that,
Catalysis as a function of pH. A pH–rate study was con-
ducted, in order to determine whether complex 1 catalyses ester
hydrolysis as anticipated. The pH–rate profile for the hydrolysis
of p-NPA (observed as the release of the p-nitrophenolate
anion) in the presence of complex 1 is reproduced in Fig. 3,
together with the profile obtained for buffer alone in the
absence of complex 1. (The reactions were run under pseudo
first-order conditions, with the catalyst in excess.) It is clear
from the plots that complex 1 catalyses the hydrolysis of p-NPA
throughout the pH range studied, with a marked increase in the
rate at pH 8. The rate enhancement afforded by the catalyst
important as the nature of the environment surrounding the
2ϩ
CA Zn ion appears to be in forming the ‘enzyme–substrate’
2ϩ
complex, a relatively ‘naked’ Zn complex is itself sufficient to
bind a substrate molecule with substantial affinity. The
elaborately-functionalised active-site of CA has presumably
evolved to afford a degree of substrate specificity and to
facilitate the formation of the ‘enzyme–substrate’ complex. A
biomimetic analogy to this is the demonstration by Zhang and
Breslow that a tailored, highly hydrophobic ester forms a
2ϩ
relatively strong complex with a Cu -based ‘artificial hydro-
lase’ equipped with appropriately-positioned hydrophobic
(
with respect to buffer alone) is 11.5 at pH 8.2. The pH (8.0) at
20
cavities.
which the sharp increase in rate occurs corresponds to the pK
a
2ϩ
of the Zn -bound water molecule which was suggested by the
titrimetric data (Fig. 2). It is therefore reasonable to propose
that the sharp increase in rate arises from the nucleo-
2ϩ
philic attack of a Zn -bound hydroxide ion on the substrate,
supporting the proposed mode of action of CA.
Michaelis–Menten kinetics. Michaelis–Menten saturation
kinetics is observed during the hydrolysis of p-NPA by complex
1
. Thus, a plot of log ν (where ν is the initial rate of reaction)
against [p-NPA] (Fig. 4) indicates the saturation of the catalyst
by the substrate. Replotting of the data in Lineweaver–Burk
form (not shown) gives a value of Km = 7.6 mM (45.0 ЊC, pH
Catalytic efficiency. The rate of hydrolysis of p-NPA as a
function of [1] is depicted in Fig. 5 as a plot of k against [1].
obs
2
8
.20, I = 0.1 M, 50% v/v CH CN–H O). To our knowledge, this
The plot is essentially linear with a slope (r = 0.993, equating to
3
2
Ϫ1 Ϫ1
is the first demonstration of Michaelis–Menten kinetics in a
carbonic anhydrase model, and the absence from complex 1
of the hydrophobic cavity and substrate-binding residue
the second-order rate constant, k ) of 0.71 M
s
(55.0 ЊC, pH
2
8.20, I = 0.1 M) in the region [1] ≤ 3 mM. The slope of the plot
approximates to zero at [1] ≥ 3 mM, which is accounted for by
the lack of solubility of 1 at >3 mM. The corresponding value
(
Thr199) of CA enables an estimation of their role in catalysis
Ϫ1 Ϫ1
17
to be made. The value of Km for the hydrolysis of p-NPA
by complex 1 is approximately 13 times greater than that of
for CA is 97 M
s
(25 ЊC, pH 7.20, I = 0.09 M), and the
enzyme is therefore a markedly more efficient catalyst of p-NPA
hydrolysis than is complex 1. To some extent, this is accounted
for by the difference in the values of Km for the two systems,
17
carbonic anhydrase (Km = 0.57 mM), and the hydrophobic
cavity and/or Thr199 residue therefore appear to increase the
J. Chem. Soc., Perkin Trans. 2, 2001, 2063–2067
2065

View Article Online
Kinetic experiments
The liberation of the p-nitrophenolate anion was observed
spectrophotometrically at 405 nm. A typical kinetic experiment
was conducted as follows. A 1 cm quartz cuvette containing
2
.5 ml of a buffered solution of the complex was temperature-
equilibrated in the spectrophotometer and then treated with an
aliquot of a solution of p-NPA in dry acetonitrile. The kinetic
run was then started and followed almost to completion
(
>6 half lives) for the determination of the observed pseudo
first-order rate constants, kobs, or to the consumption of 1–2%
of the ester for the determination of the initial rate, ν. The data
presented are the mean values of triplicate runs.
Potentiometric titrations
Protonation constants for tris(aminomethyl)ethane (with or
without one equivalent (1 mM) of Zn(ClO ) ) were obtained by
4
2
Fig. 5 Plot of kobs for the liberation of p-nitrophenolate from p-NPA
as a function of [1]. ([p-NPA] = 20 µM, pH 8.20, 55 ± 0.1 ЊC, aqueous,
titrating an aqueous solution (50 ml, 25.0 ЊC, I = 0.1 M) of
tris(aminomethyl)ethane (1 mM) and perchloric acid (3 mM)
under nitrogen with a standard aqueous solution of sodium
hydroxide (0.1 M). Each curve shown is the mean of three
independent titrations.
5
0 mM MOPS buffer, I = 0.1 M.)
although, after correcting for the difference in the temperature
at which the two values were recorded, CA remains the
more efficient catalyst by almost two orders of magnitude. The
reason for this is not clear, and further studies (both with
enzyme models and with site-directed mutagenesis) may afford
an explanation. Taking into account the difference in temper-
1
Preparation of complex 1 for H NMR analysis
A solution of complex 1 (100 ml) was prepared by dissolving
tris(aminomethyl)ethane (11.7 mg, 0.1 mmol) in water (95 ml),
and the pH was adjusted to ca. 6 by the addition of perchloric
acid (2 M). The solution was treated, with stirring, with one
equivalent of Zn(ClO ) (50 µl of a 2 M solution), and the pH
ature at which the data were obtained, the value of k for
2
Ϫ2
Ϫ1 Ϫ1
complex 1 is comparable to that for 3 (k = 4.1 × 10
pH 8.2, 25 ЊC).
M
s
at
2
9
4
2
adjusted to 8.80 by the addition of sodium hydroxide solution
(6 M). The volume of the solution was made up to 100 ml and a
check made to ensure that the pH of the solution was still ca.
Concluding remarks
These results provide an indication of the importance of active-
site functionalities to the catalytic activity of CA. They also
illustrate the advantage, in terms of catalytic efficiency, which
evolution has conferred on the enzyme. In order for a bio-
mimetic catalyst to achieve an efficiency comparable to that of
CA, the next generation of such catalysts will require additional
functionality. The systematic inclusion of moieties which model
other aspects of the enzyme architecture could help to further
8
.80. An aliquot of the solution (1 ml) was withdrawn, evapor-
ated to dryness, the residue treated with a little D O and the
2
whole evaporated to dryness once more. After a second D O
2
exchange, the residue was dissolved in 20% v/v D O in CD CN
2
3
1
(
1 ml) and the H NMR spectrum recorded. (This procedure
permits the accurate control of the pH of the solution and
avoids the need for the conversion of the pH meter reading to
the corresponding pD value.)
2ϩ
explain why simple Zn complexes are less efficient catalysts
than CA, thus bringing the prospect of outstanding ‘artificial’
enzymes a step closer.
Acknowledgements
The generous financial support of Jotun A/S is gratefully
Experimental
H NMR spectra were recorded using a Bruker AM360
acknowledged.
1
spectrometer operating at 360 MHz. UV–visible spectro-
photometric data for kinetic experiments were obtained using
either an Hewlett-Packard HP8453 or HP8452 diode-array
spectrophotometer, which was operated under PC control with
Hewlett-Packard software. Potentiometric titration and pH-stat
kinetic data were obtained using a Metrohm 736 GP Titrino
apparatus, which was operated under PC control with Metrohm
software. p-NPA was obtained from Lancaster Synthesis and
recrystallised from hexane. The recrystallised material was
stored under argon, and used to prepare 50 mM stock solutions
in dry acetonitrile, which were freshly-prepared every two
days. Good’s buffers were purchased from Sigma or Lancaster
Synthesis and were used as obtained. Zinc perchlorate hexa-
hydrate was purchased from Aldrich and used as an approxi-
mately 2 M aqueous solution, the precise concentration of
which was determined by EDTA titration. Tris(aminomethyl-
References
1
(a) Carbonic Anhydrase, eds. F. Botrè, G. Gros and B. T. Storey,
VCH, Weinheim, 1991; (b) J. N. Earnhardt and D. N. Silverman, in
Comprehensive Biological Catalysis, ed. M. Sinnott, Academic Press,
London, 1998, vol. 1, ch. 13i.
2 (a) W. N. Lipscomb and N. Sträter, Chem. Rev., 1996, 96, 2375;
(
b) D. S. Auld, Struct. Bonding, 1997, 89, 29; (c) I. Bertini and
C. Luchinat, in Bioinorganic Chemistry, eds. I. Bertini, H. B. Gray,
S. J. Lippard and J. S. Valentine, University Science Books,
Mill Valley (California), 1994, ch. 2.
3
R. D. Hancock and A. E. Martell, Adv. Inorg. Chem., 1995, 42, 89.
4 S. F. Lincoln and A. E. Merbach, Adv. Inorg. Chem., 1995, 42, 1.
5 Y. Pocker and S. Sarkanen, Adv. Enzymol., 1978, 47, 149.
6
(a) B. Greener, M. H. Moore and P. H. Walton, Chem. Commun.,
996, 27; (b) C. Kimblin, W. E. Allen and G. Parkin, J. Chem. Soc.,
1
Chem. Commun., 1995, 1813; (c) R. Alsfasser, S. Trofimenko,
A. Looney, G. Parkin and H. Vahrenkamp, Inorg. Chem., 1991,
3
0, 4098; (d ) A. Looney, G. Parkin, R. Alsfasser, M. Ruf and
21
)
ethane was prepared according to literature procedures.
H. Vahrenkamp, Angew. Chem., Int. Ed. Engl., 1992, 31, 92.
Freshly-distilled water was used throughout. Ionic strength
7 M. Ruf and H. Vahrenkamp, Chem. Ber., 1996, 129, 1025. For
an interpretation of this and related results, see M. Rombach,
C. Maurer, K. Weis, E. Keller and H. Vahrenkamp, Chem. Eur. J.,
(
I ) was maintained with NaClO4.
CAUTION: although no problems were encountered during
1
999, 5, 1013.
the use of zinc perchlorate or perchloric acid, suitable care and
precautions should be taken when handling such potentially
explosive compounds.
8
Only a few carbonic anhydrase models with catalytic activity have
been reported; examples with demonstrated esterase activity include
(a) E. Kimura, H. Hashimoto and T. Koike, J. Am. Chem. Soc.,
2
066
J. Chem. Soc., Perkin Trans. 2, 2001, 2063–2067

View Article Online
1
996, 118, 10963; (b) J. Suh, O. Han and B. Chang, J. Am. Chem.
13 (a) N. Kitajiri, T. Arishima and S. Takamoto, Nippon Kagaku
Soc., 1986, 108, 1839; (c) J. Chin and X. Zuo, J. Am. Chem. Soc.,
Zasshi, 1970, 91, 240 (Chem. Abstr., 1970, 73, 29530); (b) G.
Anderegg, Helv. Chim. Acta, 1962, 1303.
1
984, 106, 3687; (d ) D. S. Sigman and C. T. Jorgensen, J. Am. Chem.
Soc., 1972, 94, 1724.
14 H. Sigel and R. B. Martin, Chem. Soc. Rev., 1994, 23, 83.
15 A quantitative analysis of this observation using thermodynamic
data has been reported: T. Itoh, Y. Fujii, T. Tada, Y. Yoshikawa and
H. Hisada, Bull. Chem. Soc. Jpn., 1996, 69, 1265.
16 The nature of the donor atoms is also important; thus a complex
composed of a tripodal array of sulfur donors does not appear
to afford a pKa reduction: C. Kimblin, B. M. Bridgewater,
D. G. Churchill and G. Parkin, Chem. Commun., 1999, 2301.
17 Y. Pocker and J. T. Stone, J. Am. Chem. Soc., 1965, 87, 5497.
18 C. A. Fierke, J. F. Krebs and R. A. Venters, in Carbonic Anhydrase,
eds. F. Botrè, G. Gros and B. T. Storey, VCH, Weinheim, 1991,
pp. 22–35.
19 X. Xu, A. R. Lajmi and J. W. Canary, Chem. Commun., 1998, 2701.
20 B. Zhang and R. Breslow, J. Am. Chem. Soc., 1997, 119, 1676. (See
also: B. Zhang and R. Breslow, J. Am. Chem. Soc., 1998, 120, 5854).
21 (a) R. J. Geue and G. H. Searle, Aust. J. Chem., 1983, 36, 927;
(b) E. B. Fleischer, A. E. Gebala, A. Levey and P. A. Tasker,
J. Org. Chem., 1971, 36, 3042.
9
E. Kimura, T. Shiota, T. Koike, M. Shiro and M. Kodama, J. Am.
Chem. Soc., 1990, 112, 5805.
0 (a) C. Bazzicalupi, A. Bencini, A. Bencini, A. Bianchi, F. Corana,
V. Fusi, C. Giorgi, P. Paoli, P. Paoletti, B. Valtancoli and
C. Zanchini, Inorg. Chem., 1996, 35, 5540; (b) C. Bazzicalupi,
A. Bencini, A. Bianchi, V. Fusi, C. Giorgi, P. Paoletti, B. Valtancoli
and D. Zanchi, Inorg. Chem., 1997, 36, 2784.
1
1
1 For reviews of small-molecule models of a number of different
enzymes, see (a) E. Kimura and E. Kikuta, J. Biol. Inorg. Chem.,
2
(
1
000, 5, 139; (b) J. K. Bashkin, Curr. Opin. Chem. Biol., 1999, 3, 752;
c) E. Kimura, T. Koike and M. Shionoya, Struct. Bonding, 1997, 89,
; (d ) R. Breslow, Acc. Chem. Res., 1995, 28, 146; (e) E. Kimura,
Prog. Inorg. Chem., 1994, 41, 443; ( f ) K. D. Karlin, Science, 1993,
2
61, 701; (g) J. Suh, Acc. Chem. Res., 1992, 25, 273; (h) J. Chin, Acc.
Chem. Res., 1991, 24, 145.
1
2 A. Cornish-Bowden, Fundamentals of Enzyme Kinetics, Portland,
London, 1995.
J. Chem. Soc., Perkin Trans. 2, 2001, 2063–2067
2067