Paraoxon, 4-nitrophenyl phosphate and acetate are substrates of α- But not of β-, γ- And ζ-carbonic anhydrases

Article abstract of DOI:10.1016/j.bmcl.2010.08.110

Carbonic anhydrases (CAs, EC 4.2.1.1) belonging to α-, β-, γ- and ζ-classes and from various organisms, ranging from the bacteria, archaea to eukarya domains, were investigated for their esterase/phosphatase activity with 4-nitrophenyl acetate, 4-nitrophe

Full text of DOI:10.1016/j.bmcl.2010.08.110

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6208–6212
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Paraoxon, 4-nitrophenyl phosphate and acetate are substrates of a- but not of
b-,
c
- and f-carbonic anhydrases
⇑
Alessio Innocenti, Claudiu T. Supuran
Università degli Studi di Firenze, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy
Normale, 34296 Montpellier Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Carbonic anhydrases (CAs, EC 4.2.1.1) belonging to a-, b-, c- and f-classes and from various organisms, rang-
Received 31 July 2010
Revised 19 August 2010
Accepted 21 August 2010
Available online 26 August 2010
ing from the bacteria, archaea to eukarya domains, were investigated for their esterase/phosphatase activ-
ity with 4-nitrophenyl acetate, 4-nitrophenyl phosphate and paraoxon as substrates. Only -CAs showed
esterase/phosphatase activity, whereas enzymes belonging to the b-, - and f-classes were completely
devoid of such activity. Paraoxon, the metabolite of the organophosphorus insecticide parathione, was a
a
c
much better substrate for several human/murine a-CA isoforms (CA I, II and XIII), with kcat/KM in the range
Keywords:
Carbonic anhydrase
Esterase
of 2681.6–4474.9 Mꢀ1 sꢀ1, compared to 4-nitrophenyl phosphate (kcat/KM of 14.9–1374.4 Mꢀ1 sꢀ1).
Ó 2010 Elsevier Ltd. All rights reserved.
CO2 hydrase
Phosphatase
Paraoxon
Metalloenzyme
Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze a very simple but
physiologically essential reaction in all life kingdoms, the hydration
of carbon dioxide to bicarbonate and protons, with a high effi-
ciency.1,2 Although this reaction also occurs without a catalyst, it is
too slow to account for physiologic requirements in many biological
systems where this interconversion is needed (e.g., photosynthesis
in plants, algae, diatoms and some bacteria;3,4 various electrolyte-
secreting tissues in mammals;5–7 some tumors in which several
CA isoforms are involved in tumor progression and metastasis,8–10
biosynthetic processes such as gluconeogenesis, ureagenesis or lipo-
genesis,1,5,6 etc.). Indeed, CO2, bicarbonate and protons are essential
molecules/ions in many important physiologic processes in all life
kingdoms (Bacteria, Archaea, and Eukarya) throughout the phyloge-
netic tree, and high rates of their interconversion are necessary
sometimes in conditions in which the spontaneous reaction cannot
processes connected with respiration and transport of CO2/bicar-
bonate, pH and CO2 homeostasis, electrolyte secretion in a variety
of tissues/organs, biosynthetic reactions, bone resorption, calcifica-
tion, tumorigenicity, etc.1,2,5–7 Some of them are cytosolic (CA I, CA II,
CA III, CA VII and CA XIII), others are membrane-bound (CA IV, CA IX,
CA XII, CA XIV and CA XV), CA VA and CA VB are mitochondrial, and
CA VI is secreted in saliva and milk. Three acatalytic isoforms are also
known, denominated sometimes CA related proteins (CARP), that is,
CA VIII, CA X and CA XI, which are cytosolic proteins too.1,2,5–7
In addition to CO2 hydration to bicarbonate and protons, at least
the
a-CAs are known to possess other catalytic activities, such as
among others esterase activity (with 4-nitrophenyl acetate, 4-
NPA, and other activated esters).11 The related ester 4-nitrophenyl
phosphate is also a CA substrate, whereas 4-nitrophenyl sulfate is
not at all hydrolyzed by these enzymes, as recently shown by our
occur. For example, CA IX, a tumor-associated
a
-CA8–10 has a very
group.11 Indeed, in a recent study we observed that the
a-CA iso-
high turnover (of 7.5 ꢁ 107 Mꢀ1 sꢀ1) for CO2 hydration to bicarbon-
ate even at a pH of 6.5 (typical of hypoxic tumors in which this
enzyme is over-expressed) although the spontaneous, non-catalytic
CO2 hydration at this pH value is very low.10
zymes (of mammalian, human(h) or murine (m) origin), hCA I,
hCA II and mCA XIII show a good esterase activity with 4-nitro-
phenyl acetate as substrate, with second order rate constants in
the range of 753–7706 Mꢀ1 sꢀ1, being slightly less effective as
phosphatases with 4-nitrophenyl phosphate (4-NPP) 1 as substrate
(kcat/KM in the range of 14.89–1374.40 Mꢀ1 sꢀ1) and totally ineffec-
tive as sulfatases (with 4-nitrophenyl sulfate as substrate).11 As
shown in Scheme 1, hydrolysis of 1 mediated by CAs leads to the
formation of 4-nitrophenol 2.
These enzymes are also excellent examples of convergent evolu-
tion, as five distinct genetic families (
a-, b-,
c
-, d-, and f-CAs) were
discovered so far.1–4 Mammals possess 16 different
a-CA isoforms,
which are involved in many crucial physiological or pathological
It is presently not known whether enzymes belonging to other
classes than the a-CAs possess this catalytic versatility mentioned
⇑
Corresponding author. Tel.: +39 055 4573005; fax: +39 055 4573385.
E-mail address: claudiu.supuran@unifi.it (C.T. Supuran).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.110

A. Innocenti, C. T. Supuran / Bioorg. Med. Chem. Lett. 20 (2010) 6208–6212
6209
domains, are shown, for the CO2 hydration, 4-NPA hydrolysis and
4-NPP hydrolysis reactions.18–27 The CO2 hydrase activities of all
these enzymes (human (h) and murine (m) CAs belonging to the
O
P
OH
OH
O
OH
a
-class, under the form of several isoforms, such as hCA I, hCA II
and mCA XIII;11 an
-CA from the bacterium Helicobacter pylori,
hp
a
H2O
CA
a
CA,21 the b-class CA from the same organism, hpbCA;22 another
+ H3PO4
bacterial enzyme, from Brucella suis, bsCA 1,23 Cab, a b-CA from the
archaeon Methanobacterium thermoautotrophicum; ScCA,24 the
b-CA from the yeast Saccharomyces cerevisiae;25 Cam, the prototyp-
NO2
NO2
ical c
-CA from the archaeon Methanosarcina thermophila;26 as well
1: 4-NPP
2
as the cadmium/zinc-containing CA1 repeat (Cd/Zn(II)-R1) from
the marine diatom Thalassiosira weissflogii)27 are quite high, with
turnovers in the range of 8.3 ꢁ 104–1.6 ꢁ 108 Mꢀ1 sꢀ1 (Table 1).
We have included in our experiments enzymes belonging to very
diverse organisms (bacteria, archaea, diatoms, yeasts and mam-
mals) and four of the five classes (details on d-CAs are very scarce
in the literature and no enzyme from this family has been thor-
oughly characterized so far)28 in order to investigate in detail the
putative catalytic versatility of CAs for other reactions than CO2
hydration. These enzymes show medium-low CO2 hydrase activity
(mCA XIII and Cab), moderate such activity (hCA I, the two
enzymes from H. pylori, bsCA 1, ScCA and Cam) or very high CO2
hydrase activity (hCA II, Cd(II)-R1 and Zn(II)-R1). Thus, the hydrase
activity may be quite efficient irrespective whether the enzymes
O
P
OEt
OEt
O
OH
H2O
CA
+
(EtO)2P(=O)OH
NO2
NO2
3
2
CA
-AcO-
S
belong to the
data of Table 1, only the
activity, whereas for the various enzymes belonging to the b-,
- and f-CA family, such activities were not detected. The kcat/KM
a-, b-,
c- and f-CA classes. However, as seen from
O
P
OEt
OEt
a
-CAs show also esterase and phosphatase
O
O
c
values for these reactions are on the other hand orders of magni-
tude lower compared to the CO2 hydrase activity. Indeed, for
4-NPA hydrolysis the kcat/KM values ranged between 654 and
7706 Mꢀ1 sꢀ1 (the best esterase was mCA XIII which possesses
the lowest hydrase activity) whereas for the phosphate ester 1
hydrolysis, they were in the range of 38.9–1374.4 Mꢀ1 sꢀ1 (again
with isoform CA XIII the best phosphatase, Table 1). It should be
NO2
NO2
4-NPA
4
Scheme 1. Hydrolysis of the phosphate esters 4-nitrophenyl phosphate (4-NPP) 1
and paraoxon 3 to 4-nitrophenol 2. The insecticide parathion 4 is metabolized to
paraoxon 3, which we demonstrate here to be a CA substrate. 4-Nitrophenylacetate
(4-NPA) is also hydrolyzed by CAs with formation of 2 and acetate.
mentioned that the phosphatase activity of all a-CAs with 1 as sub-
strate showed a bell-shaped pH dependency with an optimal activ-
ity between pH 7.1 and 7.5. At pH values >8, this activity started to
be highly diminished probably due to the fact that the zinc-
hydroxide moiety of the enzyme has repulsive interactions with
the ester 1 which is a dianion at that pH.
above, that is, in addition to the hydration of CO2 to bicarbonate
and protons, it is unknown whether the b-,
c
-, d-, and f-CAs may
The explanation of this lack of esterase or phosphatase activity
also act as esterases, phosphatases or eventually sulfatases. On
the other hand, only 4-nitrophenyl phosphate 1 has been investi-
gated to date as a possible substrate for the phosphatase activity
of these enzymes,11 although phosphate esters are highly relevant
in a host of physiological processes (as recently reviewed by Black-
burn,12 some of the most important ones being: DNA stabilization;
the RNA world, presumably the first forms of self-replication made
possible by ribozymes; phospholipid; skeletal structures; organelle
membrane recognition mediated by phosphoinositols; energy rich
phosphates such as ATP, ADP; phosphorylation/dephosphorylation
of proteins mediated by kinases/phosphatases; second messengers
such as cGMP, cAMP, etc., just to mention the really essential
ones).12,13
for the non-
the metal ion coordination is rather similar at least between the
- and -CAs (three His residues and a water molecule/hydroxide
ion) whereas for most of the b- and all f-CAs this is accomplished
by two Cys, one His and the catalytically essential water molecule/
hydroxide ion.1–5 However, it should be mentioned that the active
site dimensions are very much different between these enzymes,
a-CAs is not straightforward, considering the fact that
a
c
with the
a-CAs possessing a wide and deep cavity of around
15 ꢁ 15 Å2,29 the b-CAs possess a channel of around 5 Å deep (much
shallower compared to the a c-CA has
-CA active site),30 whereas the
the active site even smaller, a cleft at the interface between two adja-
cent subunits of the protein homotrimer.3a–c However, the f-CA
shoes a bigger active site compared to the b- and c-CAs, of around
In this contribution we try to reply to some of the question
7 ꢁ 7 Å2.4 Maybe, the access of substrate much bulkier than CO2,
raised above, that is, whether the non-
a-CAs show catalytic versa-
such as the esters 4-NPA and 4-NPP, is very much hindered in the
tility, possessing esterase and/or phosphatase activity. We also re-
port here that paraoxon 3, an organophosphorus compound acting
as acetylcholinesterase inhibitor14,15 (being the metabolite of para-
thion 4, a widely used insecticide)16,17 and structurally rather sim-
smaller active sites of the enzymes belonging to the b-,
CA classes, whereas it may occur much easier in the wide and deep
active site of the -CAs. We have also investigated the sulfatase
activity of the enzymes shown in Table 1, with 4-nitrophenyl sulfate
as substrate, and all of them, similar to the mammalian ones inves-
tigated earlier,11 are devoid of esterase activity (data not shown).
In order to see whether other organic phosphates may act as
c- and f-
a
ilar to 4-NPP 1, is a much better
phosphate 1.
a-CA substrate compared to the
In Table 1 the kinetic parameters (as kcat/KM) for several CAs
belonging to various enzyme families ( -, b-, - and f-CA family)
and found in organisms from the bacteria, archaea or eukarya
a
c
substrates of the
a-CAs, we investigated whether paraoxon 3, an
ester structurally related to 4-NPP 1, may be hydrolyzed enzymat-

6210
A. Innocenti, C. T. Supuran / Bioorg. Med. Chem. Lett. 20 (2010) 6208–6212
Table 1
Kinetic parameters for various reactions catalyzed by CAs, at 25 °C18–20
Enzymea
Class
CO2 hydration
kcat/KM (Mꢀ1 sꢀ1
)
Ref.
4-NPA hydrolysis
4-NPP hydrolysis
hCA I
hCA II
mCA XIII
a
a
a
a
b
b
b
b
c
f
5.0 ꢁ 107
1.5 ꢁ 108
8.3 ꢁ 104
1.5 ꢁ 107
4.8 ꢁ 107
3.9 ꢁ 107
1.8 ꢁ 106
9.8 ꢁ 107
3.9 ꢁ 106
1.4 ꢁ 108
1.6 ꢁ 108
753
2607
7706
654
0
65.55
14.89
1374.4
38.9
0
0
0
0
0
11
11
11
hpa
CA
This study, 21
This study, 22
This study, 23
This study, 24
This study, 25
This study, 26
This study, 27
This study, 27
hpbCA
bsCA 1
Cab
ScCA
Cam
0
0
0
0
0
0
Cd(II)-R1
Zn(II)-R1
0
0
f
For the CO2 hydration reaction the pH was of 7.4 for the a-, c- and f-class enzymes and of 8.3 for the b-CAs (which show much lower activity at
pH <8).1–3 The CO2 hydrase activities of all these enzymes were reported earlier,11,22–27 whereas the esterase ones only for the mammalian
CAs.11 The esterase and phosphatase activities provided in the table were measured at a pH of 7.4 but many other pH values (between 5.5 and
9.0) have been investigated without measuring any catalytic enhancement in the presence of b-, c- and f-CAs.
a
h = human, m = murine isoforms; hpCA = Helicobacter pylori CA; bsCA = Brucella suis CA; Cab = Methanobacterium thermoautotrophicum
enzyme; ScCA = Saccharomyces cerevisiae CA; Cam = Methanosarcina thermophila enzyme; Cd(II)-R1 = R1 repeat of cadmium-containing CA1
from Thalassiosira weissflogii; Zn(II)-R1 = R1 repeat of zinc-containing CA1 from T. weissflogii.
ically by
a
-CAs (hCA I, hCA II and mCA XIII, Table 2). It should be
in the range of 2681.6–4474.9 Mꢀ1 sꢀ1 (Table 2). Thus, paraoxon 3
is a much better substrate compared to 4-NPP 1. Probably the pres-
ence of the two additional ethyl moieties in 3 allows a better inter-
action with the enzyme active site compared to the parent ester 1.
Indeed, it may be observed that the KM values are rather different
but of the same order of magnitude for the two esters and a partic-
ular CA isoform (except mCA XIII, for which there is a difference of
almost one order of magnitude between the KM of the two esters,
with paraoxon showing a value of 2.20 mM and phosphate 1 of
only 0.232 mM). The kcat/KM values are on the other hand very dif-
ferent when comparing the two substrates. Indeed, hCA I and hCA
II showed a very weak phosphatase activity with 4-NPP 1 as sub-
strate (turnovers of 14.89–65.55 Mꢀ1 sꢀ1), and as mentioned
above, only mCA XIII had a relevant phosphatase activity with this
ester. However, with 3 as substrate, all these enzymes behave as
rather efficient phosphatases (Table 2), and it may be thus noted
that they behave as ‘paraoxonases’. It should be mentioned that
CA XIII is the unique member of this enzyme family having a Val
in position 200.1,2 This amino acid residue (His in CA I and Thr in
CA II) is essential for the binding of substrates and inhibitors to
the CAa,1,2 and its more hydrophobic nature in CA XIII may facili-
tate the binding of hydrophobic esters such as 4-NPP and parao-
xon. Furthermore, this activity (with both phosphates as
substrates) was inhibited by the clinically used sulfonamide aceta-
zolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide), with
IC50 values in the range of 59–1050 nM (Table 2). This is a proof
that the zinc hydroxide mechanism responsible for the CO2 hy-
drase activity of these enzymes is also responsible of the esterase
mentioned that there are enzymes, named paraoxonases (PON,
EC 3.1.8.1), known to efficiently hydrolyze 3.31–33 At least three
PON isoforms are known so far in humans (h), hPON 1–hPON 3.
hPON1 is associated with high-density lipoprotein (HDL), and is a
genetically polymorphic enzyme that has the ability to hydrolyze
a wide range of organophosphates and carboxylic acid esters.31
hPON 1 active site contains a Ca(II) ion critical for catalysis (and
a second structural such ion) coordinated by five protein residues
(the side chain oxygens of Asn224, Asn270, Asn168, Asp269 and
Glu53) and by a water molecule which in deprotonated form (as
hydroxide ion) acts as nucleophile for the hydrolysis of sub-
strates.31 Another potential ligand of the catalytic metal ion is an
oxygen of the phosphate substrate which will undergo hydrolysis,
but the rate determining step of the catalytic turnover is the depro-
tonation of the calcium-coordinated water molecule, assisted by a
His–His dyad,31 which very much resembling the CA catalytic cy-
cle, in which a Zn(II) hydroxide species is the catalytically active
nucleophile, which is generated by deprotonation of zinc-coordi-
nated water assisted by a His residue from the enzyme active
site.1–3 Thus, two very different enzymes, with very diverse metal
ions in the active site share a rather similar catalytic mechanism.
This, and the fact that we showed earlier11 that some
sess phosphatase activity with 4-NPP as substrate, prompted us
to investigated paraoxon 3 as a possible substrate of CAs.
As seen from data of Table 2, paraoxon 3 is a good substrate for
a-CAs pos-
the three mammalian a-CAs investigated here, with kcat/KM values
and phosphatase activity of the
a-CAs. The phosphatase activity
of these -CAs with paraoxon as substrate showed no pH depen-
a
Table 2
dency between pH 7.1 and 8.5, probably because at these pH val-
ues there is a substantial amount of the enzyme in the zinc
hydroxide form, and paraoxon is in neutral form, unlike 4-NPP
which may be a mono- or dianion, depending on the pH in the as-
say system.
Kinetic parameters for the hydrolysis of 4-nitrophenyl phosphate (1) and paraoxon
(3) in the presence of cytosolic CA isoforms I, II and XIII, at pH 7.4 and 25 °C, and
inhibition data with acetazolamide (5-acetamido-1,3,4-thiadiazole-2-sulfonamide)
Isozymea
Substrate
kcat/KM (Mꢀ1 sꢀ1
)
KM (mM)
IC50 (nM)
hCA I
hCA I
hCA II
hCA II
mCA XIII
mCA XIII
1
3
1
3
1
3
65.55 5.2
3893.0 14.5
14.89 0.54
4474.9 58
1374.4 62
2681.6 47
0.935 0.10
1.683 0.12
2.195 0.20
1.645 0.28
0.232 0.02
2.202 0.14
330 14
338 12
In conclusion, we prove here that only CAs belonging to the
-class but not the b-, c- and f-classes show esterase/phosphatase
activity with 4-nitrophenyl acetate, 4-nitrophenyl phosphate and
paraoxon as substrates. Paraoxon, the metabolite of the organo-
phosphorus compound parathione, was a much better substrate
a
63
59
5
4
1050 76
1021 63
for several human/murine
a-CA isoforms (CA I, II and XIII), with
Concentrations of the substrates 1 and 3 ranged between 0.08 and 5 mM. The data
are provided as the mean standard deviation (from at least three different
assays).18,19
kcat/KM in the range of 2681.6–4474.9 Mꢀ1 sꢀ1
, compared to
4-nitrophenyl phosphate (kcat/KM of 14.9–1374.4 Mꢀ1 sꢀ1).
show thus a modest but significant paraoxonase activity.
a-CAs
a
h = human, m = murine isoform.

A. Innocenti, C. T. Supuran / Bioorg. Med. Chem. Lett. 20 (2010) 6208–6212
6211
substrate, at least six traces of the initial 5–10% of the reaction have been used
for determining the initial velocity. The uncatalyzed rates were determined in
the same manner and subtracted from the total observed rates. Stock solutions
of inhibitor (10 mM) were prepared in distilled-deionized water and dilutions
up to 0.01 nM were done thereafter with distilled-deionized water. Inhibitor
and enzyme solutions were preincubated together for 15 min at room
temperature prior to assay, in order to allow for the formation of the E-I
complex. The inhibition constants were obtained by non-linear least-squares
methods using PRISM 3, whereas the kinetic parameters for the uninhibited
enzymes from Lineweaver–Burk plots, as reported earlier,11 and represent the
mean from at least three different determinations.
Acknowledgments
Research from our laboratory was financed by a grant of the 7th
FP of EU (Metoxia project).
References and notes
1. (a) Supuran, C. T. Nat. Rev. Drug Disc. 2008, 7, 168; (b) Supuran, C. T. Bioorg. Med.
Chem. Lett. 2010, 20, 3467.
19. Pocker, Y.; Stone, J. T. Biochemistry 1968, 7, 2936. 4-Nitrophenyl acetate
(4-NPA) hydrolysis was monitored by measuring the absorbance at 405 nm of
2. (a) Supuran, C. T. Carbonic Anhydrases as Drug Targets—General Presentation.
In Drug Design of Zinc-Enzyme Inhibitors: Functional, Structural, and Disease
Applications; Supuran, C. T., Winum, J. Y., Eds.; Wiley: Hoboken, NJ, 2009; pp
15–38; (b) Winum, J. Y.; Rami, M.; Scozzafava, A.; Montero, J. L.; Supuran, C.
Med. Res. Rev. 2008, 28, 445; (c) Supuran, C. T.; Scozzafava, A.; Casini, A. Med.
Res. Rev. 2003, 23, 146; (d) Silverman, D. N. Biochim. Biophys. Acta 2010, 1804,
243; (e) Domsic, J. F.; Avvaru, B. S.; Kim, C. U.; Gruner, S. M.; Agbandje-
McKenna, M.; Silverman, D. N.; McKenna, R. J. Biol. Chem. 2008, 283, 30766.
3. (a) Ferry, J. F. Biochim. Biophys. Acta 2010, 1804, 374; (b) Smith, K. S.; Jakubzick,
C.; Whittam, T. S.; Ferry, J. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 15184; (c)
Zimmerman, S. A.; Tomb, J. F.; Ferry, J. G. J. Bacteriol. 2010, 192, 1353; (d)
Zimmerman, S. A.; Ferry, J. G.; Supuran, C. T. Curr. Top. Med. Chem. 2007, 7, 901;
(e) Elleuche, S.; Pöggeler, S. Microbiology 2010, 156, 23.
the chromogenic compound 4-nitrophenol
2 which is released by the
hydrolysis of the ester (extinction coefficient of 10510 Mꢀ1).11 Reactions
were performed at 25 °C in a quartz cuvette with 1 cm light-path, using a
Perkin Elmer Lambda Bio20 UV–vis spectrometer. Enzyme concentrations
were between 0.10 and 5.0 lM and substrate concentration between 0.08 and
0.37 mM. The substrate was dissolved in freshly distilled acetonitrile and
diluted with buffer (10 mM HEPES and 10 mM Tris, pH 7.4, maintaining the
ionic strength constant by addition of 0.1 M sodium sulfate, in such a way that
the final concentration of MeCN was of 5%). Kinetic parameters were
determined (in cases in which the substrate solubility allowed this, i.e.,
concentrations of 4-NPA of 0.3–5.0 mM) by fitting the data to the Michaelis–
Menten model (Eq. 1):
4. (a) Xu, Y.; Feng, L.; Jeffrey, P. D.; Shi, Y.; Morel, F. M. Nature 2008, 452, 56; (b)
Cox, E. H.; McLendon, G. L.; Morel, F. M.; Lane, T. W.; Prince, R. C.; Pickering, I. J.;
George, G. N. Biochemistry 2000, 39, 12128; (c) Lane, T. W.; Morel, F. M. Proc.
Natl. Acad. Sci. U.S.A. 2000, 97, 4627.
5. (a) Pastorekova; Parkkila, S.; Pastorek, J.; Supuran, C. T. J. Enzyme Inhib. Med.
Chem. 2004, 19, 199; (b) Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. Expert
Opin. Ther. Pat. 2006, 16, 1627; (c) Ebbesen, P.; Pettersen, E. O.; Gorr, T. A.; Jobst,
G.; Williams, K.; Kienninger, J.; Wenger, R. H.; Pastorekova, S.; Dubois, L.;
Lambin, P.; Wouters, B. G.; Supuran, C. T.; Poellinger, L.; Ratcliffe, P.; Kanopka,
A.; Görlach, A.; Gasmann, M.; Harris, A. L.; Maxwell, P.; Scozzafava, A. J. Enzyme
Inhib. Med. Chem. 2009, 24, 1.
Vo ¼ ðkcat½Eꢂ0½Sꢂ0Þ=ð½Sꢂ0 þ KM
Þ
ð1Þ
linear fit (Eq. 2) by using
ð2Þ
Otherwise, kcat/KM values were derived from
a
PRISM:11
Vo ¼ ½Eꢂ0½Sꢂ0ðkcat=KM
Þ
6. (a) Supuran, C. T. Curr. Pharm. Des. 2008, 14, 641; (b) Supuran, C. T.; Di Fiore, A.;
De Simone, G. Expert Opin. Emerg. Drugs 2008, 13, 383; (c) De Simone, G.; Di
Fiore, A.; Supuran, C. T. Curr. Pharm. Des. 2008, 14, 655; (d) Mincione, F.;
Scozzafava, A.; Supuran, C. T. Antiglaucoma Carbonic Anhydrase Inhibitors as
Ophthalomologic Drugs. In Drug Design of Zinc-Enzyme Inhibitors: Functional,
Structural, and Disease Applications; Supuran, C. T., Winum, J. Y., Eds.; Wiley:
Hoboken, NJ, 2009; pp 139–154; (e) Krungkrai, J.; Supuran, C. T. Curr. Pharm.
Des. 2008, 14, 631; (f) Borras, J.; Scozzafava, A.; Menabuoni, L.; Mincione, F.;
Briganti, F.; Mincione, G.; Supuran, C. T. Bioorg. Med. Chem. 1999, 7, 2397.
7. Alterio, V.; Hilvo, M.; Di Fiore, A.; Supuran, C. T.; Pan, P.; Parkkila, S.; Scaloni, A.;
Pastorek, J.; Pastorekova, S.; Pedone, C.; Scozzafava, A.; Monti, S. M.; De Simone,
G. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 16233.
The rates of spontaneous hydrolysis (without enzyme) were subtracted from
the enzymatic rates. Inhibition with acetazolamide has been used as a control,
being performed as described above, by titration of the enzymes with acetazol-
amide solutions in concentration ranges between 10 nM to 100 lM. IC50 repre-
sents the molarity of inhibitor producing a 50% decrease of the enzyme activity
and were determined from semilogarithmic plots of enzyme activity versus
molarity of inhibitor.11
20. Pullan, L. M.; Noltmann, E. A. Biochemistry 1985, 24, 635. The phosphatase
activity with 1 and 3 as substrates has been assayed by a variant of the method
used by Pullan and Noltmann, measuring the absorbance at 405 nm of the
8. (a) De Simone, G.; Supuran, C. T. Biochim. Biophys. Acta 2010, 1804, 404; (b)
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chromogenic group 4-nitrophenol 2 which is released according to the
reactions of Scheme 1. The assay has been performed at 25 °C in the same
buffer used for the esterase activity measurements described above, working at
substrate concentrations of 0.08–5 mM (variants of the assay have been also
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performed at pH values between 5.5–9 for the various a, b-, c- and f-CAs
investigated here, but no catalytic enhancement has been detected at any pH
tested). Kinetic parameters were determined as described above, and the rates
of spontaneous hydrolysis (without enzyme) were subtracted from the
measured enzymatic rates. Inhibition with acetazolamide has again been
used as a control, being performed as described above, by titration of the
enzymes with acetazolamide solutions in the concentration range between
11. Innocenti, A.; Scozzafava, A.; Parkkila, S.; Puccetti, L.; De Simone, G.; Supuran,
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10 nM to 100 lM. IC50 values were calculated as described above. The sulfatase
activity with 4-nitrophenyl sulfate (4-NSA) as substrate, has been investigated
in an analogous manner, and working at various pH values (in the range of
5.4–8.5, data not shown) but was totally absent with all investigated enzymes,
in all experimental conditions employed.
13. (a) Cliff, M. J.; Bowler, M. W.; Varga, A.; Marston, J. P.; Szabó, J.; Hounslow, A.
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working at the absorbance maximum of 557 nm, with 10–20 mM Hepes (pH
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7.5, for
a- and c-CAs) or Tris (pH 8.3 for b-CAs) as buffers, and 20 mM Na2SO4
(for -CAs) or 10–20 mM NaCl—for b-CAs (for maintaining constant the ionic
a
strength), following the initial rates of the CA-catalyzed CO2 hydration reaction
for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for
the determination of the kinetic parameters. For each concentration of the

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