The physiological role of mammalian CuAOs and therefore their role in the complexity of cellular action depend highly on the interaction with other biomolecules. To identify the physiological molecules acting as substrates or modulators of the enzyme activity, it is necessary to known both the structure of the enzyme and the type of forces controlling the step of molecular recognition. The specificity of these interactions depends on the van der Waals forces, hydrogen bonding, Coulomb interactions and hydrophobic effects. In particular, the dielectric constant of the microenvironment influences the extent of the Coulomb interactions and consequently also the pKa of acid groups, which can be present in the active sites of the enzymes. In this study we have combined kinetic and spectroscopic studies with the aim to estimate the dielectric constant of the hydrophobic region into the active site of bovine serum amine oxidase (BSAO). This hydrophobic region previously hypothesized by kinetic studies (1-3) and then identified by crystallographic data (4), lies at about 5-10 Angstroms from the TPQ cofactor and can interact by hydrophobic effect with the substrates and/or inhibitors of BSAO. Aliphatic monoamines with different chain length have been used as substrates to probe the “hydrophobicity” of BSAO active site by kinetic studies. Fluorescent probes have been chemically bound to TPQ and have been used as “sensors” of dielectric constant. From these data, a relative dielectric constant of about 12 was estimated for this hydrophobic zone of BSAO interacting with the apolar moiety of biomolecules. This finding on the microenvironmental dielectric properties of BSAO can explain the high pKa value found for its catalytic base (ASP 385) (5) and could be common also to the hydrophobic pocket present also in the human enzyme, VAP-1 (6-7). This behaviour should be keep in mind in future docking studies and in the design of new specific inhibitors with pharmacological purpose, in particular for the human VAP-1
On the characteristics of the hydrophobic pocket into BSAO active site
DI PAOLO, MARIA LUISA;
2006
Abstract
The physiological role of mammalian CuAOs and therefore their role in the complexity of cellular action depend highly on the interaction with other biomolecules. To identify the physiological molecules acting as substrates or modulators of the enzyme activity, it is necessary to known both the structure of the enzyme and the type of forces controlling the step of molecular recognition. The specificity of these interactions depends on the van der Waals forces, hydrogen bonding, Coulomb interactions and hydrophobic effects. In particular, the dielectric constant of the microenvironment influences the extent of the Coulomb interactions and consequently also the pKa of acid groups, which can be present in the active sites of the enzymes. In this study we have combined kinetic and spectroscopic studies with the aim to estimate the dielectric constant of the hydrophobic region into the active site of bovine serum amine oxidase (BSAO). This hydrophobic region previously hypothesized by kinetic studies (1-3) and then identified by crystallographic data (4), lies at about 5-10 Angstroms from the TPQ cofactor and can interact by hydrophobic effect with the substrates and/or inhibitors of BSAO. Aliphatic monoamines with different chain length have been used as substrates to probe the “hydrophobicity” of BSAO active site by kinetic studies. Fluorescent probes have been chemically bound to TPQ and have been used as “sensors” of dielectric constant. From these data, a relative dielectric constant of about 12 was estimated for this hydrophobic zone of BSAO interacting with the apolar moiety of biomolecules. This finding on the microenvironmental dielectric properties of BSAO can explain the high pKa value found for its catalytic base (ASP 385) (5) and could be common also to the hydrophobic pocket present also in the human enzyme, VAP-1 (6-7). This behaviour should be keep in mind in future docking studies and in the design of new specific inhibitors with pharmacological purpose, in particular for the human VAP-1Pubblicazioni consigliate
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