2270
A. Innocenti et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2267–2271
with 3 as substrate, but the sulfatase activity could not
be detected.
4. Supuran, C. T.; Conroy, C. W.; Maren, T. H. Proteins:
Struct. Funct. Genetics 1997, 27, 272.
5
. (a) Briganti, F.; Mangani, S.; Scozzafava, A.; Vernaglione,
G.; Supuran, C. T. J. Biol. Inorg. Chem. 1999, 4, 528; (b)
Guerri, A.; Briganti, F.; Scozzafava, A.; Supuran, C. T.;
Mangani, S. Biochemistry 2000, 39, 12391.
It is interesting to note that esters 2 and 3 (as sodium
salt, used in these experiments) are conjugate bases of
strong acids. Thus, probably both 2 and 3 are in anionic
form at all pH ranges in which experiments have been
performed. It is thus difficult to rationalize the different
behavior of the phosphate 2 and sulfate 3 as possible
substrates of CAs. A possible electrostatic repulsion be-
tween the oxygen sulfate atom(s) of 3 and the zinc
hydroxide species of the enzyme cannot be taken into
account, since phosphate 2 is probably also bound in an-
ionic form to the CA active site and acts as a CA sub-
strate. Obviously, acetate 1 is bound as neutral species
to the CA active site, allowing the strong nucleophilic
6
. (a) Pocker, Y.; Dickerson, D. G. Biochemistry 1968, 7,
1995; (b) Pocker, Y.; Meany, J. E. Biochemistry 1967, 6,
239.
7. (a) Pocker, Y.; Stone, J. T. Biochemistry 1967, 6, 668; (b)
Verpoorte, J. A.; Mehta, S.; Edsall, J. T. J. Biol. Chem.
1967, 242, 4221.
8
9
. Kaiser, E. T.; Lo, K. W. J. Am. Chem. Soc. 1969, 91, 4912.
. Henkart, P.; Guidotti, G.; Edsall, J. T. J. Biol. Chem.
1
968, 243, 2447.
1
1
0. (a) Register, A. M.; Koester, M. K.; Noltmann, E. A.
J. Biol. Chem. 1978, 253, 4143; (b) Pullan, L. M.;
Noltmann, E. A. Biochemistry 1985, 24, 635.
1. Kim, G.; Selengut, J.; Levine, R. L. Arch. Biochem.
Biophys. 2000, 377, 334.
12. Nishimori, I.; Minakuchi, T.; Onishi, S.; Vullo, D.; Cecchi,
A.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem.
2007, 15, 7229.
3. Lehtonen, J.; Shen, B.; Vihinen, M.; Casini, A.; Scozzaf-
ava, A.; Supuran, C. T.; Parkkila, A. K.; Saarnio, J.;
Kivela, A. J.; Waheed, A.; Sly, W. S.; Parkkila, S. J. Biol.
Chem. 2004, 279, 2719.
4. Hilvo, M.; Tolvanen, M.; Clark, A.; Shen, B.; Shah, G.
N.; Waheed, A.; Halmi, P.; H a¨ nninen, M.; H a¨ m a¨ l a¨ inen, J.
M.; Vihinen, M.; Sly, W. S.; Parkkila, S. Biochem. J. 2005,
392, 83.
15. Host, G.; Martensson, L. G.; Jonsson, B. H. Biochim.
Biophys. Acta 2006, 1764, 1601.
16. Gould, S. M.; Tawfik, D. S. Biochemistry 2005, 44, 5444.
2
+
ꢀ
(
Zn (OH) ) attack, without any electrostatic repul-
sions, being thus able to effectively hydrolyze it accord-
ing to Eq. 11 of Scheme 2.
In conclusion, we investigated in detail the esterase,
phosphatase, and sulfatase activities of three cytosolic
CA isozymes, hCA I, II, and mCA XIII with 4-nitro-
phenyl esters as substrates. These enzymes showed a
good esterase activity with 4-nitrophenyl acetate as
substrate, with second order rate constants in the
1
1
ꢀ
1
ꢀ1
, being slightly less effective
as phosphatases (kcat/KM in the range of 14.89–
range of 753–7706 M
s
ꢀ
1
ꢀ1
s ) and totally ineffective as sulfatases.
1
374.40 M
These esterase/phosphatase activities were inhibited by
sulfonamide CA inhibitors, proving that the zinc-
hydroxide mechanism responsible for the CO hydrase
1
7. Gambhir, K. K.; Ornasir, J.; Headings, V.; Bonar, A.
Biochem. Genet. 2007, 45, 431.
2
activities of these enzymes is also responsible of their
esterase/phosphatase activity. CA XIII was the most
effective esterase and phosphatase, although it showed
reduced hydrase activity as compared to CA I and II.
It is probable that CA XIII might catalyze other physi-
1
8. (a) Jarvela, S.; Parkkila, S.; Bragge, H.; Kahkonen, M.;
Parkkila, A. K.; Soini, Y.; Pastorekova, S.; Pastorek, J.;
Haapasalo, H. BMC Cancer, 2008, 8, in press.; (b)
Haapasalo, J.; Nordfors, K.; J a¨ rvel a¨ , S.; Bragge, H.;
Rantala, I.; Parkkila, A. K.; Haapasalo, H.; Parkkila, S.
Neuro Oncol. 2007, 9, 308; (c) Kummola, L.; H a¨ m a¨ l a¨ inen,
J. M.; Kivel a¨ , J.; Kivel a¨ , A. J.; Saarnio, J.; Karttunen, T.;
Parkkila, S. BMC Cancer 2005, 5, 41; (d) Leppilampi, M.;
Koistinen, P.; Savolainen, E. R.; Hannuksela, J.; Parkkila,
A. K.; Niemel a¨ , O.; Pastorekov a´ , S.; Pastorek, J.; Waheed,
A.; Sly, W. S.; Parkkila, S.; Rajaniemi, H. Clin. Cancer
Res. 2002, 8, 2240.
ological reactions than CO hydration, based on its rel-
2
evant phosphatase activity reported here.
Acknowledgment
This research was financed in part by two grants of the
1
9. Pocker, Y.; Stone, J. T. Biochemistry, 1968, 7, 2936.
Ester 1 hydrolysis was monitored by measuring the
absorbance at 405 nm of the chromogenic group 4-
nitrophenol 4 which is released according to Eq. 11,
6
(
th Framework Programme of the European Union
EUROXY and DeZnIT projects).
ꢀ
1
16
Scheme
2
(extinction coefficient of 10,510 M ).
References and notes
Reactions were performed at 25 ꢁC in a quartz cuvette
with 1 cm lightpath, using a Perkin-Elmer Lambda
Bio20 UV–vis spectrometer. Enzyme concentrations
were between 0.10 and 5.0 lM and substrate concen-
tration 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, that is, concentrations of ester
1 of 0.3–5.0 mM) by fitting the data to the Michaelis–
Menten model (Eq. 14):
1
. (a) Supuran, C. T. Nat. Rev. Drug Discov. 2008, 7, 168; (b)
Supuran, C. T.; Scozzafava, A.; Conway, J. Carbonic
Anhydrase—Its Inhibitors and Activators; CRC Press:
Boca Raton, New York, London, 2004, pp 1–363.
. (a) Pastorekova, S.; Parkkila, S.; Pastorek, J.; Supuran, C.
T. J. Enzyme Inhib. Med. Chem. 2004, 19, 199; (b)
Supuran, C. T.; Scozzafava, A.; Casini, A. Development
of sulfonamide carbonic anhydrase inhibitors. In Carbonic
Anhydrase—Its Inhibitors and Activators; Supuran, C. T.,
Scozzafava, A., Conway, J., Eds.; CRC Press: Boca
Raton, 2004; pp 67–147.
2
3
. (a) Supuran, C. T. Therapy 2007, 4, 355; (b) Supuran, C.
T. Curr. Top. Med. Chem. 2007, 7, 825; (c) Supuran, C. T.;
Scozzafava, A. Bioorg. Med. Chem. 2007, 15, 4336.
Vo ¼ ðkcat½Eꢁ ½Sꢁ Þ=ð½Sꢁ þ K
Þ
ð14Þ
0
0
0
M