Lactoperoxidase (LPO) is a mammalian redox enzyme that catalyzes the oxidation of halides, pseudo-halides and a number of aromatic substrates at the expense of hydrogen peroxide1. LPO is a large glycoprotein (612 residues, Mr = 78.5 KDa) characterised by a covalently-bound heme cofactor and a high number of disulfide bridges. It plays a complex physiological role, due to its involvement in the natural host-defence system against bacterial infections, as well as in carcinogenic mechanisms, cystic fibrosis and inflammatory processes. Thermal denaturation studies2 have shown that LPO is a highly thermostable enzyme, since it keeps its catalytic proficiency and structural cohesion up to 70°C. Such high thermal stability relies on the presence of a -helix-rich core that preserves the heme pocket from being structurally damaged. However, other factors and interactions are likely to play a role in this process and remain to be characterized. These include the ester bonds that anchor the heme group to the apoprotein, the six disulfide bridges, the electrostatic interactions and the H-bond network inside the protein. In order to address the role of these different contributions, we have investigated the effect of chemical denaturants (urea and guanidinium chloride) and pH variations on the structure and activity of LPO3. A complementary approach, based on the use of biophysical techniques (electron spin resonance, optical absorption, fluorescence emission and circular dichroism spectroscopies) and biochemical assays, was employed. Our findings suggest that a major contribution to the enzyme stability is provided by electrostatic interactions and disulfide bridges, whereas ester bonds are crucial for its functional rather than structural role.

Chemical unfolding studies on bovine lactoperoxidase highlight the structural determinants of the enzyme stability

BOSCOLO, BARBARA;GHIBAUDI, Elena Maria;
2008-01-01

Abstract

Lactoperoxidase (LPO) is a mammalian redox enzyme that catalyzes the oxidation of halides, pseudo-halides and a number of aromatic substrates at the expense of hydrogen peroxide1. LPO is a large glycoprotein (612 residues, Mr = 78.5 KDa) characterised by a covalently-bound heme cofactor and a high number of disulfide bridges. It plays a complex physiological role, due to its involvement in the natural host-defence system against bacterial infections, as well as in carcinogenic mechanisms, cystic fibrosis and inflammatory processes. Thermal denaturation studies2 have shown that LPO is a highly thermostable enzyme, since it keeps its catalytic proficiency and structural cohesion up to 70°C. Such high thermal stability relies on the presence of a -helix-rich core that preserves the heme pocket from being structurally damaged. However, other factors and interactions are likely to play a role in this process and remain to be characterized. These include the ester bonds that anchor the heme group to the apoprotein, the six disulfide bridges, the electrostatic interactions and the H-bond network inside the protein. In order to address the role of these different contributions, we have investigated the effect of chemical denaturants (urea and guanidinium chloride) and pH variations on the structure and activity of LPO3. A complementary approach, based on the use of biophysical techniques (electron spin resonance, optical absorption, fluorescence emission and circular dichroism spectroscopies) and biochemical assays, was employed. Our findings suggest that a major contribution to the enzyme stability is provided by electrostatic interactions and disulfide bridges, whereas ester bonds are crucial for its functional rather than structural role.
2nd EuCheMS Chemistry Congress: Chemistry, the global science”
Torino (Italy)
September 16-20th 2008
Proceedings of the 2nd EuCheMS Chemistry Congress
18
18
http://www.euchems2008.unito.it/site/home.asp
protein folding; protein stability; EPR spectroscopy; CD spectroscopy; fluorescence spectroscopy; bioinorganic chemistry
B. Boscolo; S. Leal; C.Salgueiro; E. Ghibaudi; C. Gomes
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/132673
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