title: Toward Understanding the Catalytic Mechanism of Human Paraoxonase 1: Site-Specific Mutagenesis at Position 192.
abstract: Human paraoxonase 1 (h-PON1) is a serum enzyme that can hydrolyze a variety of substrates. The enzyme exhibits anti-inflammatory, anti-oxidative, anti-atherogenic, anti-diabetic, anti-microbial and organophosphate-hydrolyzing activities. Thus, h-PON1 is a strong candidate for the development of therapeutic intervention against a variety conditions in human. However, the crystal structure of h-PON1 is not solved and the molecular details of how the enzyme hydrolyzes different substrates are not clear yet. Understanding the catalytic mechanism(s) of h-PON1 is important in developing the enzyme for therapeutic use. Literature suggests that R/Q polymorphism at position 192 in h-PON1 dramatically modulates the substrate specificity of the enzyme. In order to understand the role of the amino acid residue at position 192 of h-PON1 in its various hydrolytic activities, site-specific mutagenesis at position 192 was done in this study. The mutant enzymes were produced using Escherichia coli expression system and their hydrolytic activities were compared against a panel of substrates. Molecular dynamics simulation studies were employed on selected recombinant h-PON1 (rh-PON1) mutants to understand the effect of amino acid substitutions at position 192 on the structural features of the active site of the enzyme. Our results suggest that, depending on the type of substrate, presence of a particular amino acid residue at position 192 differentially alters the micro-environment of the active site of the enzyme resulting in the engagement of different subsets of amino acid residues in the binding and the processing of substrates. The result advances our understanding of the catalytic mechanism of h-PON1.
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar (Mohali) -160062, Punjab, India.
authors.forename: Geetika
authors.initials: G
authors.lastname: Aggarwal
authors.affiliation: Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar (Mohali) -160062, Punjab, India.
authors.forename: Rameshwar
authors.initials: R
authors.lastname: Prajapati
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar (Mohali) -160062, Punjab, India.
authors.forename: Rajan K
authors.initials: RK
authors.lastname: Tripathy
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar (Mohali) -160062, Punjab, India.
authors.forename: Priyanka
authors.initials: P
authors.lastname: Bajaj
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar (Mohali) -160062, Punjab, India.
authors.forename: A R Satvik
authors.initials: AR
authors.lastname: Iyengar
authors.affiliation: Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar (Mohali) -160062, Punjab, India.
authors.forename: Abhay T
authors.initials: AT
authors.lastname: Sangamwar
authors.affiliation: Department of Biotechnology, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar (Mohali) -160062, Punjab, India.
authors.forename: Abhay H
authors.initials: AH
authors.lastname: Pande
mesh_terms: D000595:Amino Acid Sequence; D043303:Aryldialkylphosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D055162:Biocatalysis; D002118:Calcium / Q000378:metabolism; D020134:Catalytic Domain; D057075:Enzyme Assays; D006801:Humans; D006860:Hydrogen Bonding; D006868:Hydrolysis; D007700:Kinetics; D007783:Lactones / Q000378:metabolism; D008024:Ligands; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000302:isolation & purification; D010755:Organophosphates / Q000378:metabolism; D016415:Sequence Alignment; D040681:Structural Homology, Protein
chemical_list: D007783:Lactones; D008024:Ligands; D050505:Mutant Proteins; D010755:Organophosphates; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human; D002118:Calcium
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