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.

Evaluation of Association Between Q192R and L55M Genetic Polymorphisms of PON1 and Serum Paraoxonase-1 Activity in Healthy Individuals, a Meta-Analysis

“…Several association studies indicating that serum paraoxonase activity was associated with numerous diseases [3,11,12,[17][18][19]. Site directed mutagenesis revealed that the missense substitutions Q192R (rs662) and L55M (rs854560) were associated with alteration in paraoxonase activity.…”

“…Using site directed mutagenesis at position 192 of PON1 indicated that the mutant enzyme (192R) has 1.7-fold more activity compared with the wild type (192Q) protein [17]. It is well established that several genetic variations at the PON1 promoter region are correlated with the gene expression level [12,37].…”

“…It has been reported that the rs662 and rs854560 PON1 genetic variations are associated with serum paraoxonase activity [2,12]. According to site directed mutagenesis at position 192 of the PON1, the mutant 192R enzyme compared to the 192Q wild-type, show an enhanced (about 1.7-fold) paraoxonase activity [17].…”

“…Serum paraoxonase activity shows highly variability between individuals [10- 12,17]. Numerous studies indicated that the paraoxonase activity in healthy participants significantly is associated with the Q912R and L55M polymorphisms .…”

Paraoxonase 3: Structure and Its Role in Pathophysiology of Coronary Artery Disease

“…All PONs utilize a catalytic calcium ion, which functions as an oxy-anionto stabilize substrate and reaction states. Additionally, this enzyme active site employs two histidine residues (His115 and 134) involved in proton transfers, a glutamic acid(Glu53) to stabilize reactive hydrogens, and an asparagine(Asn168) to stabilize transition states and intermediates in the active site [31,38,62,63,68]. The exact mechanism is still a subject of further research and it is suggested that the His115 residue is not necessary for the lactonase and arylesterase activity of the enzyme [69].…”