G. Díaz-Díaz et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 332–336
335
Table 2
the reaction medium or in the active centre of CPO, which is also
in accordance with the cooperativity index value. So we can con-
clude that homotropic cooperativity takes place in the oxidative
Turnover numbers (min−1) for catalytic degradation of TCP.
Enzyme/catalyst
Turnover
Reaction conditions
References
(min−1
)
CPO
DHP
1200
Potassium phosphate
pH 3a
[15]
[30]
198
Sodium acetate pH 5.4a
4. Conclusion
6120
3300
4080
Sodium acetate pH 5.4a
Sodium citrate pH 3.0a
Sodium acetate pH 4.0a
[30]
[31]
[31]
HRP
The kinetics of the oxidative dehalogenation of TCP catalyzed by
CPO was studied. A sigmoidal profile was observed for the first time
when the effect of the substrate concentration on the initial reac-
tion rate was studied. A cooperative index of 1.7 0.2 was obtained,
which is more likely to be due to the formation of ꢀ–ꢀ dimers at
the binding site that to the presence of enzyme isoforms, since the
enzyme used had a high degree of purity. The kinetic parameters
n, vmax, kcat, the pseudo-Michaelis constant and the catalytic effi-
ciency were estimated, and we can conclude that CPO is one of the
most efficient catalysts for TCP dehalogenation following HRP, even
when sigmoidal kinetics are observed.
Mb
0.74
300
1200
Potassium phosphate
pH 7.0b
[32]
[33]
[34]
LiP
Sodium acetate pH
2.5–3.0a
FeTPPS
Sodium citrate/sodium
phosphate pH 3.0c
CPO: chloroperoxidase from Caldariomyces fumago, DHP: dehaloperoxidase from
Amphitrite ornata, HRP: horseradish peroxidase, Mb: myoglobin from horse heart,
LiP: lignin peroxidase from Phanerochaete chrysosporium, TPPS: meso-tetrakis(4-
sulfonatophenyl)porphyrinato.
a
100 M TCP, 400 M H2O2, 0.1 M enzyme, and 100 mM buffer.
150 M TCP, 65 M H2O2, 60 M enzyme, and 100 mM buffer.
10 mM TCP, 50 mM H2O2, 0.03 mM FeTPPS, and 100 mM buffer.
b
c
Acknowledgments
(6 × 108 M−1 min−1) and pentachlorophenol (2.3 × 107 M−1 min−1
)
Financial support was provided by the Spanish Government
(CTQ2008-02429/BQU)andthe European SocialFund(ESF). G. Díaz-
Díaz also thanks the Spanish Ministerio de Educación y Ciencia and
the ESF for a FPI grant. MS measurements were carried out by Mass
Spectrometry Unit from the University of Oviedo.
where no cooperative effects were observed [20]. Moreover,
similar catalytic efficiencies were observed for 2,2ꢀ-azino-
bis(3-ethylbenzothiazoline-6-sulfonic acid) (1.2 × 107 M−1 min−1
)
[4], anthracene (5.4 × 107 M−1 min−1), naphthalene (6.8 × 107
M
kinetics [29].
The kinetic characterization was completed with the constants
concentration (10−5 or 10−4 M), and the kinetic parameters were
recorded in Table 3.
[1] M. Sundaramoorthy, J. Terner, T.L. Poulos, Structure 3 (1995) 1367–1377.
[2] M. Sundaramoorthy, J. Terner, T.L. Poulos, Chem. Biol. 5 (1998) 461–473.
[3] H. Chen, H. Hirao, E. Derat, I. Schlichting, S. Shaik, J. Phys. Chem. B 112 (2008)
9490–9500.
[4] K.M. Manoj, L.P. Hager, Biochemistry 47 (2008) 2997–3003.
[5] G.L. Ketters, P.F. Hollenberg, Arch. Biochem. Biophys. 233 (1984) 315–321.
[6] A. Zaks, D.R. Dodds, J. Am. Chem. Soc. 117 (1995) 10419–10424.
[7] S. Hu, L.P. Hager, J. Am. Chem. Soc. 121 (1999) 872–873.
[8] F.J. Lakner, K.P. Cain, L.P. Hager, J. Am. Chem. Soc. 119 (1997) 443–444.
[9] S. Colonna, N. Gaggero, G. Carrera, P. Pasta, Chem. Commun. 5 (1997) 439–440.
[10] M.P.J. van Deurzen, F. van Rantwijk, R.A. Sheldon, J. Mol. Catal. B 2 (1996) 33–42.
[11] D.R. Doerge, M.D. Corbett, Chem. Res. Toxicol. 4 (1991) 556–560.
[12] M. Filizola, G.H. Loew, J. Am. Chem. Soc. 122 (2000) 3599–3605.
[13] W.D. Woggon, H.A. Wagenknecht, C. Claude, J. Inorg. Biochem. 83 (2001)
289–300.
[14] L.P. Hager, D.R. Morris, F.S. Brown, H.J. Eberwein, J. Biol. Chem. 241 (1996) 769.
[15] R.L. Osborne, G.M. Raner, L.P. Hager, J.H. Dawson, J. Am. Chem. Soc. 128 (2006)
1036–1037.
[16] R.L. Osborne, M.K. Coggins, J. Terner, J.H. Dawson, J. Am. Chem. Soc. 129 (2007)
14838–14839.
[17] C.E. La Rotta, E. D’Elia, E.P.S. Bon, Electron. J. Biotechnol. 10 (2007) 24–30.
[18] C.E. La Rotta, E.P.S. Bon, Appl. Biochem. Biotechnol. 98–100 (2002) 191–203.
[19] C.E. La Rotta, S. Lütz, A. Liesse, E.S. Bon, Enzyme Microb. Technol. 37 (2005)
582–588.
[20] A. Longoria, R. Tinoco, R. Vázquez-Duhalt, Chemosphere 72 (2008) 485–490.
[21] C.D. Murphy, Biotechnol. Lett. 29 (2007) 45–49.
[22] G. Lente, J.H. Espenson, Chem. Commun. 10 (2003) 1162–1163.
[23] K.M. Manoj, Biochim. Biophys. Acta 1764 (2006) 1325–1339.
[24] E. Laurenti, E. Ghibaudi, S. Ardissone, R.P. Ferrari, J. Inorg. Biochem. 95 (2003)
171–176.
[25] R.L. Osborne, M.K. Coggins, G.M. Raner, M. Walla, J.H. Dawson, Biochemistry 48
(2009) 4231–4328.
forms and distinct ligand binding sites [36–38]. However, evidence
of simultaneous binding of multiple ligands to a single active site
has been demonstrated [39–41] and this last hypothesis is widely
accepted [35]. Since CPO shares structural features with P450 [1,2],
the existence of two substrate molecules at the CPO active site, and
a theoretical study of substrate docking into the enzyme active site
[29,42] demonstrates that a ꢀ–ꢀ dimer is energetically favored
against the binding of monomeric substrate. Furthermore, Manoj
and Hager [4] corroborate the existence of at least two different
sites in CPO for carrying out oxidative reactions. Since the CPO
preparation used in this work is almost pure (Rz = 1.5), the sigmoidal
kinetics cannot be explained in terms of different isoforms of the
way that the binding of one TCP molecule facilitates the successive
binding of one more TCP molecule (n = 1.7 0.2). Assuming that
substrates that posses aromatic rings in their structure are able
to form ꢀ–ꢀ dimers [29,42], two TCP molecules could interact in
[26] S. Franzen, L.B. Gilvey, J.L. Belyea, Biochim. Biophys. Acta 1774 (2007) 121–130.
[27] I.H. Segel, Enzyme Kinetics, Behaviour and Analysis of Rapid Equilibrium and
Steady-State Systems, John Wiley & Sons, USA, 1993, pp. 346–375.
[28] E. Torres, J. Aburto, Arch. Biochem. Biophys. 437 (2005) 224–232.
[29] J. Aburto, J. Correa-Basurto, E. Torres, Arch. Biochem. Biophys. 480 (2008)
33–40.
[30] R.L. Osborne, L.D. Taylor, K.P. Han, J.H. Dawson, Biochem. Biophys. Res. Com-
mun. 324 (2004) 1194–1198.
[31] R.P. Ferrari, E. Laurenti, F. Trotta, J. Biol. Inorg. Chem. 4 (1999) 232–237.
[32] R.L. Osborne, M.K. Coggins, M. Walla, J.H. Dawson, Biochemistry 46 (2007)
9823–9829.
Table 3
Kinetic parameters obtained by varying H2O2 concentration at pH 5.0 and a fixed
concentration of TCP (10−5 or 10−4 M).
Kinetic parameters
[TCP] = 10−5
2.2 0.4
M
[TCP] = 10−4
1.3 0.1
M
n
vmax (M min−1
)
(4.9 0.1) × 10−6
(1.8 0.2) × 10−5
139 26
kcat (min−1
Ks* (M)
)
38
6
(6.2 0.4) × 10−6
(6 1) × 106
0.991
(2.3 0.4) × 10−3
(6 2) × 104
0.999
[33] K.H. Hammel, P.J. Tardone, Biochemistry 27 (1988) 6563–6568.
[34] G. Labat, J.L. Seris, B. Meunier, Angew. Chem. Int. Ed. Engl. 29 (1990) 1471–1473.
[35] W.M. Atkins, Annu. Rev. Pharmacol. Toxicol. 45 (2005) 291–310.
kcat/Ks* (M−1 min−1
)
R2