α-carbonic anhydrases possess thioesterase activity

Article abstract of DOI:10.1021/ml500470b

The α-carbonic anhydrases (CAs, EC 4.2.1.1) show catalytic versatility acting as esterases with carboxylic, sulfonic, and phosphate esters. Here we prove by kinetic, spectroscopic, and MS studies that they also possess thioesterase activity with a dithiocarbamate ester as a substrate (PhSO₂NHCSSMe). Its CA-mediated hydrolysis leads to benzenesulfonamide, methyl mercaptan, and COS. The CA thioesterase activity may be useful for designing prodrug enzyme inhibitors, whereas some CA isoforms may use this activity for modulating physiologic/pathologic processes, which are possibly amenable to drug discovery of agents with multiple mechanisms of action.

Full text of DOI:10.1021/ml500470b

Letter
pubs.acs.org/acsmedchemlett
α‑Carbonic Anhydrases Possess Thioesterase Activity
Muhammet Tanc,†,‡ Fabrizio Carta,‡ Andrea Scozzafava,‡ and Claudiu T. Supuran*,†,‡
†Laboratorio di Chimica Bioinorganica, Universita
Sesto Fiorentino, Florence, Italy
̀
degli Studi di Firenze, Polo Scientifico, Room 188, Via della Lastruccia 3, 50019
‡NEUROFARBA Department, Sezione di Scienze Farmaceutiche e Nutraceutiche, Universita
50019 Sesto Fiorentino, Florence, Italy
̀
degli Studi di Firenze, Via Ugo Schiff 6,
S
* Supporting Information
ABSTRACT: The α-carbonic anhydrases (CAs, EC 4.2.1.1)
show catalytic versatility acting as esterases with carboxylic,
sulfonic, and phosphate esters. Here we prove by kinetic,
spectroscopic, and MS studies that they also possess
thioesterase activity with a dithiocarbamate ester as a substrate
(PhSO2NHCSSMe). Its CA-mediated hydrolysis leads to
benzenesulfonamide, methyl mercaptan, and COS. The CA
thioesterase activity may be useful for designing prodrug
enzyme inhibitors, whereas some CA isoforms may use this
activity for modulating physiologic/pathologic processes, which
are possibly amenable to drug discovery of agents with multiple
mechanisms of action.
KEYWORDS: Carbonic anhydrase, thioesterase, inhibitors, prodrugs
he carbonic anhydrases are metalloenzymes, which
catalyze the interconversion between CO2 and bicar-
bonate, being involved in a multitude of physiologic and
pathologic processes.1−6 Many enzymes belonging to the six
genetic families known so far (the α-, β-, γ-, δ-, ζ-, and η-
classes) are in fact drug targets, with some of their inhibitors in
clinical use for decades.1−3,7−9
In addition to the physiologic reaction, the α-CAs, which are
found in many organisms, from bacteria to humans,1−4 also
show catalytic versatility, acting as esterases with carboxylic,
sulfonic, and phosphate esters,10−14 as well as hydration
properties toward substrates that are structurally similar to
CO2, such as COS, CS2, cyanamide, and aldehydes.15−18 It is
not known whether other such catalyzed reactions, apart CO2
hydration, have some physiologic relevance.
bovine (b) and human (h) red cell CA (bCA, hCA II) as well
as the Co(II)-substituted derivative, bCo(II)CA, which has an
electronic spectrum highly sensitive to the environment around
the Co(II) ion.22
T
The compound we used to demonstrate the esterase activity
was the methyl phenylsulfonylcarbamodithioate (1), prepared
as described in the Supporting Information (see also Scheme
1). The inhibition of the hCA II with compound 1 is shown in
Figure 1, with benzenesulfonamide 3 as standard inhibitor.23−25
A stopped-flow assay with CO2 as substrate has been
employed to measure the inhibitory power of compounds 1
and 3.23 As seen from Figure 1, the dithiocarbamate ester 1
showed a time-dependent inhibition constant (Ki) against hCA
II, which steeply varied with the time of incubation between the
It is interesting to note that at this moment the possible
thioesterase activity of the CAs has not been investigated,
although considering the similar mechanism of reaction for the
hydrolysis of carboxylic and thiocarboxylic esters, such an
activity may be expected. Indeed, recently we have discovered
that coumarins are a new class of CA inhibitors (CAIs).19−21
Their mechanism of inhibition is distinct of that of all classes of
CAIs investigated earlier, as they are first hydrolyzed (at the
lactone ring) to 2-hydroxy-cinnamic acids, through the esterase
activity of the enzyme. The phenolic carboxylic acid formed in
this way then binds at the entrance of the active site cavity.19
Such a type of inhibition was then observed also for monocyclic
lactones as well as for the thiocoumarins and thiolactones.20,21
On the basis of such data we had the suspicion that CAs may
also possess thioesterase activity. Here we demonstrate this new
catalytic activity of the α-CAs by using as model enzyme of the
Scheme 1. Suggested Mechanism for Thioesterase Activity of
hCA II on Dithiocarbamate Ester 1
Received: November 15, 2014
Accepted: January 17, 2015
© XXXX American Chemical Society
A
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Letter
Figure 1. Inhibition constant (Ki) change for methyl phenyl-
sulfonylcarbamodithioate 1 versus time, incubated with hCA II for
1−18 h(s), blue; Ki change for 1 versus time in HEPES buffer (without
enzyme), red. *Errors in the range of 10 of the reported values, from
three different stopped-flow assays.
Figure 3. bCo(II)CA (6 μM, blue line), in HEPES; bCo(II)CA with
methyl phenylsulfonylcarbamodithioate 1 (1:2) (red); after 8 h
(green); after 16 h (pink). In all experiments, 20 nM HEPES (pH 7.5)
as buffer and 20 mM Na2SO4 were used.
enzyme and compound 1. The Ki continued to decrease until
an incubation period of 12 h was reached, suggesting that the
compound undergoes a transformation in the presence of
enzyme. It should be noted that compound 1 incubated in the
same conditions with the buffer but without enzyme showed
the same inhibition constant over time, suggesting that the
hydrolytic process is indeed mediated by CA and not by other
nucleophiles present in the buffer (Figure 1).
enzyme (data not shown), but the spectra typical of an adduct
of bCo(II)CA with a sulfonamide appear after 8 h of incubation
of the enzyme and the thioester (Figure 3).
The final evidence that the thioester 1 is hydrolyzed to
benzenesulfonamide 3 and COS came from the mass spectra
(MS) experiments shown in Figure 4. After incubation of
compound 1 with hCA II for 16 h, the characteristic peak of
COS was observed at 63.9, together with the benzenesulfona-
mide peak at 156 m/z (Figure 4). The peak of unreacted 1 was
also observed at 246 m/z.
Figures 2 and 3 show the electronic spectra of bCo(II)CA
complexed with benzenesulfonamide 3 (Figure 2) and
Figure 2. bCo(II)CA (6 μM) in HEPES buffer (blue); bCo(II)CA
with benzenesulfonamide 3 (1:2) (red); after 10 min (green); after 15
min (pink). Twenty nanomolar HEPES (pH 7.5) as buffer, and 20
mM Na2SO4 (for maintaining constant ionic strength) used in all
experiments.
compound 1 (Figure 3) versus time, respectively. We have
chosen the dithiocarbamate ester 1 in order to eventually
generate a primary aromatic sulfonamide by hydrolysis to its
thioester moiety. It may be seen that the bCo(II)CAII has a
typical electronic spectrum, with maxima at 520 and 560 nm,
and the sulfonamide 3 adduct of the Co(II)-substituted enzyme
is immediately formed, having again quite characteristic features
and absorption maxima at 518 and 574 nm (Figure 2).17 These
spectra are typical of Co(II) in tetrahedral geometry, as the
water coordinated to the metal ion was substituted by the
sulfonamidate anion of the inhibitor.22 It should be also noted
that the spectrum of the Co(II)-substituted enzyme with the
dithiocarbamate ester 1 is initially very similar to the spectra of
pure bCo(II)CA shown in Figure 2. No initial changes of the
spectrum are seen when 1 is added to the Co(II)-substituted
Figure 4. Mass spectra (MS) analyses of methyl phenylsulfonylcarba-
modithioate 1 (0.1 mM) incubated with hCA II (0.1 μM) for 16 h.
(A) Negative ESI fragmentation of compound 1 to give phenyl-
sulfonamide 3. (B) In positive ESI mode, COS+ is detected.
B
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The proposed mechanism for the thioesterase activity of CA
is shown in Scheme 1. The nucleophilic attack of the hydroxide
ion bound to the Zn(II) (or Co(II) ions) from the enzyme
active site to the CS carbon atom of the thioester
functionality gives a tetrahedral intermediate (not shown),
which loses methyl mercaptan 2 and leads to the formation of a
presumably unstable monothiocarbamic acid intermediate,
which collapses to benzenesulfonamide 3 and COS 4.
human fatty acid synthase possesses a thioesterase domain that
can be targeted by small molecule inhibitors leading to relevant
antitumor effects.27 As many α-CA isoforms are present in
tumors,28−30 we cannot exclude that, in addition to their pH
regulating effects, some CAs may use their thioesterase activity
for modulating physiologic/pathologic processes, which might
be amenable to drug discovery of antitumor agents with
multiple mechanisms of action.30
It is worth mentioning that due to the particular substrate
chosen, which by hydrolysis leads to benzenesulfonamide, the
thioesterase activity of the enzyme is strongly inhibited by one
of the reaction products, i.e., the primary sulfonamide. The
reason why we chose this particular thioester was dictated by
the fact that we wanted to use kinetics, electronic spectroscopy,
and MS for demonstrating this new enzymatic activity of the
CAs. Indeed, the spectra of the Co(II)-substituted CA with
sulfonamides are highly characteristic, proving clearly the
interaction between the inhibitor and the metal ion from the
enzyme active site. In addition, the high resolution MS allowed
us to evidence the formed products in the hydrolysis of
thioester 1 mediated by CA, which prompted us to propose the
reaction mechanism of the thioesterase reaction.
ASSOCIATED CONTENT
* Supporting Information
Synthesis and NMR/MS spectra of compound 1 and the
experimental details for the kinetic, spectroscopic, and MS
experiments. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Tel: +39-055-457-3005. Fax: +39-055-4573385. E-mail:
claudiu.supuran@unifi.it.
■
Funding
This research was financed by the EU FP7Marie Curie ITN
project Dynano. We are grateful to Dr. Riccardo Romoli for his
technical support with mass spectrometric experiments.
Thioesterases are abundant enzymes in all life kingdoms,
being involved in crucial physiologic processes. There are 27
different clans of such enzymes, which hydrolyze the thioester
bond between a carbonyl moiety and a sulfur atom.26 For 15
out of 27 such groups, the substrates are thioesters of coenzyme
A (CoA), whereas two contain acyl carrier proteins (ACP), four
have glutathione or its derivatives as substrates, one has
ubiquitin, and two such enzyme families possess other diverse
substrates.26,27 One important aspect of this enzyme super-
family is that they are not metalloenzymes except for one
family, the hydroxyglutathione hydrolases (glyoxalases II),
which are zinc enzymes with a metallo-β-lactamase fold.26
Indeed, in all other thioesterase clans known to date the
thioester hydrolysis is achieved by a catalytic dyad/triad
normally comprising a nucleophilic amino acid−histidine−
acidic amino acid sequence. Some of the most common
catalytic residues known are Asp-Gln-Thr; Cys-His-Asn, Asn-
Arg; Ser-His-Asp, etc.26 In all cases in which the catalytic
mechanism has been investigated, the nucleophile from the
catalytic triad/dyad attacks the thioester bond, with formation
in some cases of acyl-enzyme intermediates, whereas the
remaining amino acids from the triad stabilize the collapsing
tetrahedral intermediate.26 Hydroxyglutathione hydrolase has
two Zn(II) ions at the active site coordinated by seven His and
Asp residues and a bridging water molecule, which presumably
acts as nucleophile in the thioesterase reaction. Thus, the CAs
for which we have proved such an activity here are the only
other thioesterases acting by means of a metal hydroxide
mechanism, but in contrast to hydroxyglutathione hydrolase,
they do not use dinuclear metal centers. Our main conclusion is
thus that we prove that CAs belonging to the α-class possess
significant thioesterase activity and act by a new mechanism of
thioester hydrolysis compared to all other clans of thioesterases
known so far. The metal hydroxide from the CA active site is
the strong nucleophile able to attack the thiocarbonyl carbon
atom, leading to the hydrolysis of the thioester functionality. It
is unclear at this moment whether this new catalytic activity of
the CAs may have physiologic significance, but considering the
very high number of biologically crucial thioesters (e.g., the
CoA esters, ACP esters, glutathione derivatives, etc.), this may
not be excluded. In fact, very recently it has been reported that
Notes
The authors declare no competing financial interest.
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