Structure/function analyses of human serum paraoxonase (HuPON1) mutants designed from a DFPase-like homology model

Human serum paraoxonase (HuPON1) is a calcium-dependent enzyme that hydrolyzes esters, including organophosphates and lactones, and exhibits anti-atherogenic properties. A few amino acids have been shown to be essential for the enzyme's arylesterase and organophosphatase activities. Until very recently, a three-dimensional model was not available for HuPON1, so functional roles have not been assigned to those residues. Based on sequence-structure alignment studies, we have folded the amino acid sequence of HuPON1 onto the sixfold beta-propeller structure of squid diisopropylfluorophosphatase (DFPase). We tested the validity of this homology model by circular dichroism (CD) spectroscopy and site-directed mutagenesis. Consistent with predictions from the homology model, CD data indicated that the structural composition of purified HuPON1 consists mainly of beta-sheets. Mutants of HuPON1 were assayed for enzymatic activity against phenyl acetate and paraoxon. Substitution of residues predicted to be important for substrate binding (L69, H134, F222, and C284), calcium ion coordination (D54, N168, N224, and D269), and catalytic mechanism of HuPON1 (H285) led to enzyme inactivation. Mutants F222Y and H115W exhibited substrate-binding selectivity towards phenyl acetate and paraoxon, respectively. The homology model presented here is very similar to the recently obtained PON1 crystal structure, and has allowed identification of several residues within the enzyme active site.     

Structure-activity relationship on human serum paraoxonase (PON1) using substrate analogues and inhibitors

Substrate analogues based on the parent compounds paraoxon and phenyl acetate were tested on human serum paraoxonase (PON1) to explore the active site of the enzyme. Replacement of the nitro group of paraoxon with an amine or hydrogen, as well as electronic changes to the parent compound, converted these analogues into inhibitors. Introduction of either electron-withdrawing or donating groups onto phenyl acetate resulted in reduction in their rate of hydrolysis by PON1.     

Enhanced stereoselective hydrolysis of toxic organophosphates by directly evolved variants of mammalian serum paraoxonase

We addressed the ability of various organophosphorus (OP) hydrolases to catalytically scavenge toxic OP nerve agents. Mammalian paraoxonase (PON1) was found to be more active than Pseudomonas diminuta OP hydrolase (OPH) and squid O,O-di-isopropyl fluorophosphatase (DFPase) in detoxifying cyclosarin (O-cyclohexyl methylphosphonofluoridate) and soman (O-pinacolyl methylphosphonofluoridate). Subsequently, nine directly evolved PON1 variants, selected for increased hydrolytic rates with a fluorogenic diethylphosphate ester, were tested for detoxification of cyclosarin, soman, O-isopropyl-O-(p-nitrophenyl) methyl phosphonate (IMP-pNP), DFP, and chlorpyrifos-oxon (ChPo). Detoxification rates were determined by temporal acetylcholinesterase inhibition by residual nonhydrolyzed OP. As stereoisomers of cyclosarin and soman differ significantly in their acetylcholinesterase-inhibiting potency, we actually measured the hydrolysis of the more toxic stereoisomers. Cyclosarin detoxification was approximately 10-fold faster with PON1 mutants V346A and L69V. V346A also exhibited fourfold and sevenfold faster hydrolysis of DFP and ChPo, respectively, compared with wild-type, and ninefold higher activity towards soman. L69V exhibited 100-fold faster hydrolysis of DFP than the wild-type. The active-site mutant H115W exhibited 270-380-fold enhancement toward hydrolysis of the P-S bond in parathiol, a phosphorothiolate analog of parathion. This study identifies three key positions in PON1 that affect OP hydrolysis, Leu69, Val346 and His115, and several amino-acid replacements that significantly enhance the hydrolysis of toxic OPs. GC/pulsed flame photometer detector analysis, compared with assay of residual acetylcholinesterase inhibition, displayed stereoselective hydrolysis of cyclosarin, soman, and IMP-pNP, indicating that PON1 is less active toward the more toxic optical isomers.     

Direct detection of stereospecific soman hydrolysis by wild-type human serum paraoxonase

Human serum paraoxonase 1 (HuPON1; EC 3.1.8.1) is a calcium-dependent six-fold beta-propeller enzyme that has been shown to hydrolyze an array of substrates, including organophosphorus (OP) chemical warfare nerve agents. Although recent efforts utilizing site-directed mutagenesis have demonstrated specific residues (such as Phe222 and His115) to be important in determining the specificity of OP substrate binding and hydrolysis, little effort has focused on the substrate stereospecificity of the enzyme; different stereoisomers of OPs can differ in their toxicity by several orders of magnitude. For example, the C+/-P- isomers of the chemical warfare agent soman (GD) are known to be more toxic by three orders of magnitude. In this study, the catalytic activity of HuPON1 towards each of the four chiral isomers of GD was measured simultaneously via chiral GC/MS. The catalytic efficiency (k(cat)/K(m)) of the wild-type enzyme for the various stereoisomers was determined by a simultaneous solution of hydrolysis kinetics for each isomer. Derived k(cat)/K(m) values ranged from 625 to 4130 mm(-1).min(-1), with isomers being hydrolyzed in the order of preference C+P+ > C-P+ > C+P- > C-P-. The results indicate that HuPON1 hydrolysis of GD is stereoselective; substrate stereospecificity should be considered in future efforts to enhance the OPase activity of this and other candidate bioscavenger enzymes.     

In silico analyses of substrate interactions with human serum paraoxonase 1

Human paraoxonase (HuPON1) is a serum enzyme that exhibits a broad spectrum of hydrolytic activities, including the hydrolysis of various organophosphates, esters, and recently identified lactone substrates. Despite intensive site-directed mutagenesis and other biological studies, the structural basis for the specificity of substrate interactions of HuPON1 remains elusive. In this study, we apply homology modeling, docking, and molecular dynamic (MD) simulations to probe the binding interactions of HuPON1 with representative substrates. The results suggest that the active site of HuPON1 is characterized by two distinct binding regions: the hydrophobic binding site for arylesters/lactones, and the paraoxon binding site for phosphotriesters. The unique binding modes proposed for each type of substrate reveal a number of key residues governing substrate specificity. The polymorphic residue R/Q192 interacts with the leaving group of paraoxon, suggesting it plays an important role in the proper positioning of this substrate in the active site. MD simulations of the optimal binding complexes show that residue Y71 undergoes an "open-closed" conformational change upon ligand binding, and forms strong interactions with substrates. Further binding free energy calculations and residual decomposition give a more refined molecular view of the energetics and origin of HuPON1/substrate interactions. These studies provide a theoretical model of substrate binding and specificity associated with wild type and mutant forms of HuPON1, which can be applied in the rational design of HuPON1 variants as bioscavengers with enhanced catalytic activity.     

Computational characterization of how the VX nerve agent binds human serum paraoxonase 1

Human serum paraoxonase 1 (HuPON1) is an enzyme that can hydrolyze various chemical warfare nerve agents including VX. A previous study has suggested that increasing HuPON1’s VX hydrolysis activity one to two orders of magnitude would make the enzyme an effective countermeasure for in vivo use against VX. This study helps facilitate further engineering of HuPON1 for enhanced VX-hydrolase activity by computationally characterizing HuPON1’s tertiary structure and how HuPON1 binds VX. HuPON1’s structure is first predicted through two homology modeling procedures. Docking is then performed using four separate methods, and the stability of each bound conformation is analyzed through molecular dynamics and solvated interaction energy calculations. The results show that VX’s lone oxygen atom has a strong preference for forming a direct electrostatic interaction with HuPON1’s active site calcium ion. Various HuPON1 residues are also detected that are in close proximity to VX and are therefore potential targets for future mutagenesis studies. These include E53, H115, N168, F222, N224, L240, D269, I291, F292, and V346. Additionally, D183 was found to have a predicted pKa near physiological pH. Given D183’s location in HuPON1’s active site, this residue could potentially act as a proton donor or accepter during hydrolysis. The results from the binding simulations also indicate that steered molecular dynamics can potentially be used to obtain accurate binding predictions even when starting with a closed conformation of a protein’s binding or active site.     

Differential hydrolysis of homocysteine thiolactone by purified human serum (192)Q and (192)R PON1 isoenzymes

Human serum paraoxonase 1 (PON1) is a HDL-associated enzyme that catalyzes the hydrolysis of a variety of aromatic carboxylic acid esters and several organophosphates. Recently it has been suggested that a physiological substrate of serum PON1 is homocysteine thiolactone which is a putative risk factor in atherosclerosis. In this study, human (192)Q and (192)R PON1 isoenzymes were purified from the respective phenotype human serum, using a protocol consisting of ammonium sulfate precipitation and four chromatography steps: gel filtration, ion-exchange, non-specific affinity, and a second ion-exchange. Using paraoxon as substrate, overall purification fold was found as 742 for (192)R PON1 and 590 for (192)Q PON1. The final purified enzymes were shown as single protein bands close to 45kDa on SDS-PAGE and confirmed by Western blot. Substrate kinetics were studied with phenyl acetate, paraoxon and homocysteine thiolactone. Both PON1 isoenzymes showed mixed type inhibition with phenyl acetate. K(m) values of (192)Q and (192)R PON1 for homocysteine thiolactone were 23.5mM and 22.6mM respectively. For (192)R PON1, the V(max) was 2.5-fold and k(cat)/K(m) was 2.6-fold higher than those for (192)Q PON1 when homocysteine thiolactone is used as substrate. The present data suggest that defining (192)Q and (192)R PON1 isoforms could be a good predictor and prognostic marker in the cardiovascular risk assessment.     

Preferential inhibition of paraoxonase activity of human paraoxonase 1 by negatively charged lipids

To determine the causes responsible for a preferential decrease of paraoxonase activity, which has been observed in the serum of patients with cardiovascular diseases, the inactivation or inhibition of paraoxonase 1 (PON1) by various endogenous factors was examined using paraoxon or phenyl acetate as a substrate. When purified PON1 was incubated with various endogenous oxidants or aldehydes, they failed to cause a preferential reduction of paraoxonase activity, suggesting no participation of the inactivation mechanism in the preferential loss of paraoxonase activity. Next, when we examined the inhibition of PON1 activity by endogenous lipids, monoenoic acids such as palmitoleic acid or oleic acid inhibited paraoxonase activity preferentially, in contrast to a parallel inhibition of both activities by polyunsaturated or saturated acids. Noteworthy, oleoylglycine inhibited paraoxonase activity, but not arylesterase activity, complying with the selective inhibition of paraoxonase activity. Moreover, such a selective inhibition of paraoxonase activity was also expressed by lysophosphatidylglycerol or lysophosphatidylinositol, but not by lysophosphatidylserine or lysophosphatidylcholine, indicating the importance of the type of head group. Furthermore, such a preferential or selective inhibition of paraoxonase activity was also observed with PON1 associated with HDL or plasma. These data suggest that some negatively charged lipids may correspond to factors causing the preferential inhibition of paraoxonase activity of PON1.     

Interaction of checkpoint kinase 1 and the X-linked inhibitor of apoptosis during mitosis

Senescence marker protein-30 (SMP30) was originally identified as a novel protein in the rat liver, the expression of which decreases androgen-independently with aging. We have now characterized a unique property of SMP30, the hydrolysis of diisopropyl phosphorofluoridate (DFP), which is similar to the chemical warfare nerve agents sarine, soman and tabun. Hydrolysis of DFP was stimulated equally well by 1 mM MgCl₂, MnCl₂ or CoCl₂, to a lesser extent by 1 mM CdCl₂ but not at all by 1 mM CaCl₂. No ⁴⁵Ca²⁺-binding activity was detected for purified SMP30, suggesting that SMP30 is not a calcium-binding protein, as others previously stated. Despite the sequence similarity between SMP30 and a serum paraoxonase (PON), the inability of SMP30 to hydrolyze PON-specific substrates such as paraoxon, dihydrocoumarin, γ-nonalactone, and δ-dodecanolactone indicate that SMP30 is distinct from the PON family. We previously established SMP30 knockout mice and have now tested DFPase activity in their livers. The livers from wild-type mice contained readily detectable DFPase activity, whereas no such enzyme activity was found in livers from SMP30 knockout mice. Moreover, the hepatocytes of SMP30 knockout mice were far more susceptible to DFP-induced cytotoxicity than those from the wild-type. These results indicate that SMP30 is a unique DFP hydrolyzing enzyme in the liver and has an important detoxification effect on DFP. Consequently, a reduction of SMP30 expression might account for the age-associated deterioration of cellular functions and enhanced susceptibility to harmful stimuli in aged tissue.     

Homology modeling of human serum paraoxonase1 and its molecular interaction studies with aspirin and cefazolin

Human serum paraoxonase1 (HuPON1) belongs to the family of A-esterases (EC.3.1.8.1). It is associated with HDL particle and prevents atherosclerosis by cleaving lipid hydroperoxides and other proatherogenic molecules of oxidized low density lipoproteins (LDL). Since the precise structure of HuPON1 is not yet available, the structure-function relationship between HuPON1 and activators/inhibitors is still unknown. Therefore, a theoretical model of HuPON1 was generated using homology modelling and precise molecular interactions of an activator aspirin and an inhibitor cefazolin with PON1 were studied using Autodock software. The ligand binding residues were found to be similar to the predicted active site residues. Both cefazolin and aspirin were found to dock in the vicinity of the predicted active sites of PON1; cefazolin bound at residues N166, S193 and Y71, while aspirin at residues N309, I310 and L311. Binding region in the PON1 by prediction (3D2GO server) and docking studies provide useful insight into mechanism of substrate and inhibitor binding to the enzyme active site.     

Activity of paraoxonase 1 (PON1) and its relationship to markers of lipoprotein oxidation in healthy Slovaks

Low-density lipoproteins (LDLs), when modified by free radicals derived from artery wall cells, induce atherosclerosis. In contrast to oxidized LDL (ox-LDL), high-density lipoproteins (HDLs) are able to prevent atherosclerosis through a protein with antioxidant properties, paraoxonase 1 (PON1). The purpose of this study was to explore the association between the activity of HDL-associated PON1 and circulating ox-LDL as well as to investigate the relationship between ox-LDL and parameters of lipid profile in thirty Slovaks aged 21-73 years because recent studies have presented controversial results concerning PON1 and its role in LDL oxidation. For determination of circulating ox-LDL sandwich ELISA was used and other lipid parameters were determined by routine laboratory analyses. PON1 activities were assayed by two synthetic substrates - paraoxon and phenyl acetate. Lipid peroxides were determined spectrophotometrically. Of the lipid parameters examined, ox-LDL level correlated positively with total (P < 0.0001) and LDL-cholesterol (P < 0.001). Triacylglycerols (TAG) (P < 0.001), lipid peroxides (P < 0.01) and atherogenic index (AI = total cholesterol/HDL) (P < 0.0001) were also strongly correlated with ox-LDL. No inverse relationships were observed between ox-LDL and HDL-cholesterol or arylesterase/paraoxonase activities of PON1. Furthermore, it was found that ox-LDL (P < 0.01) and lipid peroxides (P < 0.05) were significantly higher in men than in women. PON1 arylesterase activity was marginally affected by sex. The results of this study suggest that the anti-atherogenic properties of HDLs are not directly related to their total concentration and that PON1 activity determined towards synthetic compounds (paraoxon and phenyl acetate) reflects no association with markers of oxidative stress. Furthermore, it follows from our results that men are more susceptible to developing atherosclerosis compared to women.     

Docking and molecular dynamics simulation studies on glycation-induced conformational changes of human paraoxonase 1

Human paraoxonase 1 (huPON1) is a calcium-dependent esterase responsible for hydrolysis of a wide variety of substrates including organophosphates, esters, lactones, and paraoxon. Although its natural substrate is unknown, the action of PON as an antioxidant is well documented. Because recent reports have suggested glycation may induce reduced PON activity in diabetes, we investigated the structural features of huPON1 and its glycated mutant by template-based modeling, docking, and molecular dynamics (MD) simulations. Our results corroborated the importance of the His115–His134 dyad in both the lactonase and paraoxonase activity of huPON1. Structural alterations in the glycated model reflected weak interactions between the docked substrate and the active site cleft. We also used MD simulation to gain insight into glycation-induced conformational changes of huPON1 and the implication of this on depleted enzymatic activity. The catalytic calcium found on the surface interacts with the side chain oxygen of residues, including Asn224, Asn270, Asn168, Asp269, and Glu53, and this interaction with the respective residues undergoes minor displacement on glycation. The root-mean-square fluctuation had high motional flexibility in the non-glycated model whereas the conformation of the glycated structure was comparatively stable. Our findings emphasize the consequence of glycation-induced alterations and their effect on overall enzymatic activity.     

The determination of Q192R polymorphism of paraoxonase 1 by using non-toxic substrate p-nitrophenylacetate

CONTEXT:

The human serum paraoxonase 1 (PON1) is calcium-dependent esterase and associates with the high density serum lipoproteins. PON1 plays a major role in oxidation of high density lipoprotein and low density lipoprotein and prevention of atherogenesis in coronary heart disease. PON1Q and R allele hydrolyses number of substrates like paraoxon (PO) (diethyl p-nitrophenyl phosphate) and phenylacetate.

AIMS:

The aim of the study is to the determination of Q192R polymorphism of PON1 by using non-toxic substrate p-nitrophenylacetate and compares it with the phenotype determined by using PO as substrate.

MATERIALS AND METHODS:

The study group consists of 60 healthy normal patients. Paraoxonase activity was measured using the procedure described by Eckerson (Reference method) and for phenotyping; the ratio of hydrolysis of PO in the presence of 1 M NaCl (salt-stimulated PON1, SALT) to the hydrolysis of phenylacetate (PA) is calculated. In new method (Haagen et al.) arylesterase activity measured using p-nitrophenylacetate and for phenotyping arylesterase, the ratio of inhibition of enzymatic hydrolysis of p-nitrophenylacetate (substrate) by phenyl acetate to non-inhibited hydrolysis of p-nitrophenylacetate (inhibited arylesterase activity (IA-IA₀)/non-inhibited arylesterase activity (NIA).

RESULTS:

It was found that paraoxonase activity is trimodally distributed in both the methods. There is no significant difference in the distribution of PON1 phenotypes of both reference method and new method being frequencies 0.946 and 0.376 respectively and there was no significant difference for phenotypic polymorphism for an individual by both methods (χ²= 0.15 and P = 0.9262).

CONCLUSION:

The Q192R polymorphism of PON1 by using non-toxic substrate p-nitrophenylacetate showed trimodal distribution of QQ (homozygous), QR (heterozygous), and RR (homozygous) phenotype and it is comparable with reference method. This method can be used for PON1 phenotype in different pathological and complex disease conditions.     

Spectrophotometric assays for the enzymatic hydrolysis of the active metabolites of chlorpyrifos and parathion by plasma paraoxonase/arylesterase

Human serum plasma paraoxonase/arylesterase exhibits a genetic polymorphism for the hydrolysis of paraoxon. One allelic form of the enzyme hydrolyzes paraoxon slowly with a low turnover number and the other(s) hydrolyzes paraoxon rapidly with a high turnover number. Chlorpyrifos-oxon, the active metabolite of the insecticide chlorpyrifos (Dursban), is also hydrolyzed by plasma arylesterase/paraoxonase. A specific assay for measuring hydrolysis of this compound is described. This assay is not subject to interference by the esterase activity of serum albumin. The Km for chlorpyrifos-oxon hydrolysis was 75 microM. Hydrolysis was inhibited by phenyl acetate, EDTA, and organic solvents. Enzyme activity required calcium ions and was stimulated by sodium chloride. Hydrolysis was optimized by using methanol instead of acetone to dissolve substrate. Unlike the multimodal distribution of paraoxonase, the distribution of chlorpyrifos-oxonase activity failed to show clear multimodality. An improvement in the assay for hydrolysis of paraoxon by plasma arylesterase/paraoxonase was achieved by elimination of organic solvents. Plotting chlorpyrifos-oxonase activity vs paraoxonase activity for a human population using the new assay conditions provided an excellent resolution of low activity homozygotes from heterozygotes for this allele. A greater than 40-fold difference in rates of chlorpyrifosoxon hydrolysis observed between rat (low activity) and rabbit sera (high activity) correlated well with the reported large differences in LD50 values for chlorpyrifos in these two animals, consistent with an important role of serum paraoxonase in detoxification of organophosphorus pesticides in vivo.     

Characterization of human paraoxonase 1 variants suggest that His residues at 115 and 134 positions are not always needed for the lactonase/arylesterase activities of the enzyme

Human paraoxonase 1 (h‐PON1) hydrolyzes variety of substrates and the hydrolytic activities of enzyme can be broadly grouped into three categories; arylesterase, phosphotriesterase, and lactonase. Current models of the catalytic mechanism of h‐PON1 suggest that catalytic residues H115 and H134 mediate the lactonase and arylesterase activities of the enzyme. H‐PON1 is a strong candidate for the development of catalytic bioscavenger for organophosphate poisoning in humans. Recently, Gupta et al. (Nat. Chem. Biol. 2011. 7, 120) identified amino acid substitutions that significantly increased the activity of chimeric‐PON1 variant (4E9) against some organophosphate nerve agents. In this study we have examined the effect of these (L69G/S111T/H115W/H134R/R192K/F222S/T332S) and other substitutions (H115W/H134R and H115W/H134R/R192K) on the hydrolytic activities of recombinant h‐PON1 (rh‐PON1) variants. Our results show that the substitutions resulted in a significant increase in the organophosphatase activity of all the three variants of rh‐PON1 enzyme while had a variable effect on the lactonase/arylesterase activities. The results suggest that H residues at positions 115 and 134 are not always needed for the lactonase/arylesterase activities of h‐PON1 and force a reconsideration of the current model(s) of the catalytic mechanism of h‐PON1.     

Indications that paraoxonase-1 contributes to plasma high density lipoprotein levels in familial hypercholesterolemia

HDL-associated paraoxonase type 1 (PON1) can protect LDL and HDL against oxidative modification in vitro and therefore may protect against cardiovascular disease. We investigated the effects of PON1 levels, activity, and genetic variation on high density lipoprotein-cholesterol (HDL-C) levels, circulating oxidized LDL (OxLDL), subclinical inflammation [high-sensitive C-reactive protein (Hs-CRP)], and carotid atherosclerosis. PON1 genotypes (L55M, Q192R, -107C/T, -162A/G, -824G/A, and -907G/C) were determined in 302 patients with familial hypercholesterolemia. PON1 activity was monitored by the hydrolysis rate of paraoxon, diazoxon, and phenyl acetate. PON1 levels, OxLDL, and Hs-CRP were determined using an immunoassay. The genetic variants of PON1 that were associated with high levels and activity of the enzyme were associated with higher HDL-C levels (P values for trend: 0.008, 0.020, 0.042, and 0.037 for L55M, Q192R, -107C/T, and -907G/C, respectively). In addition to the PON1 genotype, there was also a positive correlation between PON1 levels and activity and HDL-C (PON1 levels: r = 0.37, P < 0.001; paraoxonase activity: r = 0.23, P = 0.01; diazoxonase activity: r = 0.29, P < 0.001; arylesterase activity: r = 0.19, P = 0.03). Our observations support the hypothesis that both PON1 levels and activity preserve HDL-C in plasma.     

VX hydrolysis by human serum paraoxonase 1: a comparison of experimental and computational results

Human Serum paraoxonase 1 (HuPON1) is an enzyme that has been shown to hydrolyze a variety of chemicals including the nerve agent VX. While wildtype HuPON1 does not exhibit sufficient activity against VX to be used as an in vivo countermeasure, it has been suggested that increasing HuPON1's organophosphorous hydrolase activity by one or two orders of magnitude would make the enzyme suitable for this purpose. The binding interaction between HuPON1 and VX has recently been modeled, but the mechanism for VX hydrolysis is still unknown. In this study, we created a transition state model for VX hydrolysis (VX(ts)) in water using quantum mechanical/molecular mechanical simulations, and docked the transition state model to 22 experimentally characterized HuPON1 variants using AutoDock Vina. The HuPON1-VX(ts) complexes were grouped by reaction mechanism using a novel clustering procedure. The average Vina interaction energies for different clusters were compared to the experimentally determined activities of HuPON1 variants to determine which computational procedures best predict how well HuPON1 variants will hydrolyze VX. The analysis showed that only conformations which have the attacking hydroxyl group of VX(ts) coordinated by the sidechain oxygen of D269 have a significant correlation with experimental results. The results from this study can be used for further characterization of how HuPON1 hydrolyzes VX and design of HuPON1 variants with increased activity against VX.     

Differences in amino acid residues in the binding pockets dictate substrate specificities of mouse senescence marker protein-30, human paraoxonase1, and squid diisopropylfluorophosphatase

Senescence marker protein-30 (SMP-30) is a candidate enzyme that can function as a catalytic bioscavenger of organophosphorus (OP) nerve agents. We purified SMP-30 from mouse (Mo) liver and compared its hydrolytic activity towards various esters, lactones, and G-type nerve agents with that of human paraoxonase1 (Hu PON1) and squid diisopropylfluorophosphatase (DFPase). All three enzymes contain one or two metal ions in their active sites and fold into six-bladed β-propeller structures. While Hu PON1 hydrolyzed a variety of lactones, the only lactone that was a substrate for Mo SMP-30 was d-(+)-gluconic acid δ-lactone. Squid DFPase was much more efficient at hydrolyzing DFP and G-type nerve agents as compared to Mo SMP-30 or Hu PON1. The K(m) values for DFP were in the following order: Mo SMP-30>Hu PON1>squid DFPase, suggesting that the efficiency of DFP hydrolysis may be related to its binding in the active sites of these enzymes. Thus, homology modeling and docking were used to simulate the binding of DFP and selected δ-lactones in the active sites of Hu SMP-30, Hu PON1, and squid DFPase. Results from molecular modeling studies suggest that differences in metal-ligand coordinations, the hydrophobicity of the binding pockets, and limited space in the binding pocket due to the presence of a loop, are responsible for substrate specificities of these enzymes.     

Rate-determining step of butyrylcholinesterase-catalyzed hydrolysis of benzoylcholine and benzoylthiocholine. Volumetric study of wild-type and D70G mutant behavior.

The rate-limiting step for hydrolysis of the positively charged oxoester benzoylcholine (BzCh) by human butyrylcholinesterase (BuChE) is deacylation (k(3)), whereas it is acylation (k(2)) for hydrolysis of the homologous thioester benzoylthiocholine (BzSCh). Steady-state hydrolysis of BzCh and BzSCh by wild-type BuChE and its peripheral anionic site mutant D70G was investigated at different hydrostatic pressures, which allowed determination of volume changes associated with substrate binding, and the activation volumes for the chemical steps. A differential nonlinear pressure-dependence of the catalytic parameters for hydrolysis of both substrates by both enzymes was shown. Nonlinearity of the plots may be explained in terms of compressibility changes or rate-limiting changes. To distinguish between these two possibilities, enzyme phosphorylation by diisopropylfluorophosphate (DFP) in the presence of substrate (BzSCh) under pressure was studied. There was no pressure dependence of volume changes for DFP binding or for phosphorylation of either wild-type or D70G. Analysis of the pressure dependence for steady-state hydrolysis of substrates, and for phosphorylation by DFP provided evidence that no enzyme compressibility changes occurred during the catalyzed reactions. Thus, the nonlinear pressure dependence of substrate hydrolysis reflects changes in the rate-limiting step with pressure. Change in rate-determining step occurred at a pressure of 100 MPa for hydrolysis of BzCh by wild-type and at 75 MPa for D70G. For hydrolysis of BzSCh the change occurred at higher pressures because k(2) < k(3) at atmospheric pressure for this substrate. Elementary volume change contributions upon initial binding, productive binding, acylation and deacylation were calculated from the pressure differentiation of kinetic constants. This analysis shed light on the molecular events taking place along the hydrolysis pathways of BzCh and BzSCh by wild-type BuChE and the D70G mutant. In addition, volume change differences between wild-type and D70G provided new evidence that residue D70 in the peripheral site controls hydration of the active site gorge and the dynamics of the water molecule network during catalysis. Finally, a steady-state kinetic study of the oxyanion hole mutant (G117H) showed that substitution of the ethereal sulfur for oxygen in the substrate alters the final adjustment of substrate in the active site and stabilization of the acylation transition state.     

PON1 paraoxonase activity is reduced during HDL oxidation and is an indicator of HDL antioxidant capacity

The aim of this study was to investigate the effect of HDL oxidation on PON1 paraoxonase activity. Also, we were interested in investigating the mechanism by which PON1 could be inactivated and the correlation between its enzymatic activity and the antioxidant properties of HDL. Three different oxidation systems were used for the HDL oxidation: (1) oxidation induced by THP1 cells, (2) oxidation induced by copper ions at a concentration 10 microM, and (3) oxidation induced by *OH and O2.- oxygen free radicals produced by gamma-radiolysis. HDL oxidation was followed by the measurement of lipid peroxide formation, and PON1 activity was determined by measuring the rate of paraoxon hydrolysis. Our results show that HDL oxidation is accompanied by a reduction in the PON1 paraoxonase activity. The extent of PON1 inactivation depends both on the extent of HDL oxidation and on the oxidation system used. The rates of HDL oxidation and PON1 inactivation were significantly correlated (r = 0.93, p < 0.0054). Our results show that oxidized HDL loses its protective effect toward LDL oxidation. The antioxidant action of HDL towards LDL oxidation and the degradation of PON1 paraoxonase activity were significantly correlated (r = 0.95, p < 0.04).