Introduction

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Paraoxonase (PON1¹; EC 3.1.8.1; formerly EC 3.1.1.2) is a calcium-dependent serum enzyme belonging to the class of A-esterases (Aldridge, 1953), and it is closely associated with the high-density lipoprotein complex (Kitchen et al., 1973; Don et al., 1975; Mackness et al., 1985). PON1 is one member of a multigene family that includes at least two other genes in humans and mice (Primo-Parmo et al., 1996). Human PON1 has two common polymorphic sites: one at amino acid 55 (leucine or methionine) that results in some quantitative differences in enzyme concentration (Blatter Garin et al., 1997); and another at residue 192 (glutamine or arginine, Q/R, respectively) that accounts for marked qualitative differences in the two isozymes (Playfer et al., 1976; Smolen et al., 1991; Humbert et al., 1993). Previous studies showed that PON1 possessed hydrolytic activity with organophosphates such as paraoxon, sarin, soman, and tabun (Reiner et al., 1989; Baillie et al., 1993; Davies et al., 1996; Reiner, 1999). Paraoxonase activities of individual human serum samples showed a bimodal distribution (Playfer et al., 1976). It was later determined that the Q/R polymorphism accounted for this distribution pattern with the Q-type individuals representing the lower activity mode, and the QR heterozygotes and R homozygotes accounting for the higher activity group (Smolen et al., 1991; Humbert et al., 1993). This Q/R polymorphism presumably affects an individual's response to several toxic substrates [e.g., PON1 type R is much more effective in hydrolyzing paraoxon than PON1 type Q (Smolen et al., 1991)]. This polymorphism is being studied for its allelic association with a number of diseases such as cardiovascular disease (Ruiz et al., 1995; Serrato and Marian, 1995; Antikainen et al., 1996; Herrmann et al., 1996; Suehiro et al., 1996), carotid atherosclerosis (Schmidt et al., 1998), parkinsonism (Kondo and Yamamoto, 1998), and Alzheimer's disease (Kalman et al., 1999).

PON1 was proven to possess both arylesterase and organophosphatase activities (Sorenson et al., 1995). Some ester substrates, such as phenyl acetate, are hydrolyzed by the PON1 Q and R isozymes at approximately equivalent rates, whereas most organophosphates are hydrolyzed at different rates by the isozymes. These differences in isozymic properties allowed our laboratory to phenotype serum samples by dividing the organophosphatase activity in the presence of 1 M NaCl (with paraoxon as the substrate) by the arylesterase activity (with phenylacetate as the substrate) (Eckerson et al., 1983). Phenotyping serum samples using these simple assays aided in the purification of the two PON1 isozymes (Gan et al., 1991), which led to subsequent studies of their catalytic activities with a number of compounds.

Until recently, little additional information concerning PON1's substrate specificity beyond the structure-activity analysis described by Augustinsson and Ekedahl in 1962 has been provided. By studying substrates related to phenylacetate Augustinsson and Ekedahl concluded that a carbon-carbon double bond adjacent to the ester group was required for an ester to be hydrolyzed by PON1. In a recent abstract, we reported a new class of substrates by describing the observed hydrolysis of a large number of lactones by purified human and rabbit serum PON1. Such an activity has already been described in human and rat serum. In 1965, Roth and Giarman described the calcium-dependent lactonase activity in rat serum with -butyrolactone and -valerolactone (Roth and Giarman, 1965; Roth et al., 1967). Fishbein and Bessman (1966a,b) partially purified and characterized a similar lactonase in human blood that also required calcium and hydrolyzed -butyrolactone. They also observed that this enzyme catalyzed the reverse reaction (i.e., lactone formation) at a lower pH. Neither of these groups related this lactonase activity to serum arylesterase/paraoxonase.

During the preparation of this manuscript, an article was published describing the hydrolyses of cyclic carbonate ester compounds (Biggadike et al., 2000) by human serum. Another recent article described the hydrolysis of homocysteine thiolactone (HTL) (Jakubowski, 2000) by human serum. Both studies attributed the respective hydrolytic activities to PON1. In this article we extend our characterization of the hydrolysis of lactone and cyclic carbonate ester substrates by purified PON1 and compare the Q and R isozymes. This finding has further implications regarding the metabolism of lactone drug substrates and the substrate specificity of the other PON family members, PON2 and PON3.

PON1 esterase and organophosphatase activities have been well characterized over the past 40 years to include data on substrate structure-activity profiles, kinetics, the affects of lipoproteins and phospholipids on its hydrolytic activities, the enzyme's affinity for and requirement of specific divalent cations, the effects of salts on activities and the amino acid residues required for its activity. In this article we investigate several of these properties in relation to this newly identified lactonase activity and describe a general overview of PON1's basic enzymatic characteristics with this class of substrates.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion References

Chemicals. -Caprolactone and -caprolactam were purchased from Acros Organics USA (Fair Lawn, NJ). Phenylacetate, paraoxon, and all other lactones, thiolactones, lactams, and reagents were purchased from Sigma-Aldridge (Milwaukee, WI). Mevastatin (Compactin) and spironolactone were obtained from Sigma (St. Louis, MO); simvastatin (Zocor) and lovastatin (Mevacor) were obtained from Merck and Co. (West Point, PA) and extracted from the tablet formulations. The cyclic carbonate ester compound KB-R4899 (sodium 4-[(5-methyl-2-oxo-1,3-dioxol-4-yl) methylthio] benzenesulfonate 1/2 hydrate) was obtained from Kanebo, Ltd (Osaka, Japan).

Phenotyping and Purification of Human PON1 Types Q and R. Individual human plasma/serum was phenotyped for the PON1 Q192R polymorphism by dividing the paraoxonase activity in the presence of 1 M NaCl by the arylesterase activity (with phenyl acetate as the substrate) as described (Eckerson et al., 1983). Units of activity are defined as micromoles (arylesterase) or nanomoles (paraoxonase) of substrate hydrolyzed per minute. Human PON1 types Q and R were purified from outdated citrate plasma (obtained from the University of Michigan blood bank) as previously described (Gan et al., 1991) and modified (Kuo and La Du, 1995). The average purified PON1 preparation contains the pooled sera from three to five previously phenotyped individuals.

UV Spectrophotometric Assay. UV spectrophotometry was used to quantify the hydrolysis of lactones exhibiting reasonable absorbance differences between substrate and product, particularly for aromatic lactones. In a typical experiment the cuvette contained 1.0 mM substrate in 50 mM Tris/HCl, pH 8.0, in a total volume of 2.0 ml. The reaction was initiated by the addition of enzyme, and the increases in absorbance at 270 nm (for dihydrocoumarin), 274 nm (for 2-coumaranone), and 290 nm (for homogentisic acid lactone) were recorded. Differences in molar extinction coefficients of substrate and product were used to calculate the rate of hydrolysis. These are 876, 1295, and 816 M¹ cm¹ for dihydrocoumarin, 2-coumaranone, and homogentisic acid lactone, respectively.

Phenol Red Assay. Another general assay procedure was used for substrates with inadequate spectral differences between substrate and their corresponding hydrolysis products, but with sufficient solubility to achieve millimolar concentrations. This assay used the pH indicator dye phenol red to follow hydrogen ion release from carboxylic acid formation, the product of lactone and carbonate ester hydrolysis. The method is a modification of one developed by Sharp and Rosenberry (1982) as outlined previously (Billecke et al., 1999). Briefly, a substrate solution containing 0.004% (106 µM) phenol red, 0.005% bovine serum albumin, 2.0 mM HEPES, pH 8.0, and 1.0 mM CaCl₂ with substrate was prepared. Upon addition of enzyme, serum, or recombinant expression medium, the increase in absorbance at 422 nm was monitored and recorded. Slope values (in units of change in absorbance per minute) were multiplied by a rate factor (1900 units/ml) derived from a standard acid titration curve and divided by the sample volume (in microliters). Units were defined as the number of micromoles of acid produced (i.e., substrate hydrolyzed) per unit time.

Thiolactone Hydrolysis Assay. Thiolactone hydrolysis was measured using Ellman's procedure for monitoring the accumulation of free sulfhydryl groups via coupling with 5,5'-dithio-bis-2-nitrobenzoic acid (Ellman et al., 1961).

HPLC Analysis of Hydrolysis Products. The hydrolyses of spironolactone and the statin lactones (mevastatin, lovastatin, and simvastatin) were analyzed by HPLC using a Beckman System Gold high-performance liquid chromatograph with a model 126 programmable solvent module, a model 168 diode array detector set at 238 nm, a model 7125 rheodyne manual injector valve with a 20-µl loop, and a Beckman ODS Ultrasphere column (C₁₈, 250 × 4.6 mm, 5 µm). In a final volume of 1 ml, 50 µl of enzyme solution and 10 µl of substrate solution in methanol (0.5 mg/ml) were incubated at 25°C in 25 mM Tris/HCl (pH 7.6) and 1 mM CaCl₂. Aliquots (100 µl) were removed at specified times and added to acetonitrile (100 µl), mixed with a vortex, and centrifuged for 1 min at maximum speed (Beckman Microfuge). The supernatants were poured into new tubes, capped, and stored on ice until HPLC analysis. Samples were eluted isocratically at a flow rate of 1.0 ml/min with a mobile phase consisting of the following: A = acetic acid/acetonitrile/water (2:249:249) and B = acetonitrile, in A/B ratios of 70:30, 50:50, 45:55, and 40:60 for spironolactone, mevastatin, lovastatin, and simvastatin, respectively. Under the above conditions the retention times for the carboxylic acid formed and the lactone substrate were as follows: spironolactone (3.1 and 5.5 min), mevastatin (4.5 and 6.4 min), lovastatin (4.4 and 5.6 min), and simvastatin (4.8 and 6.6 min). Response factors for the acid products were calculated from the peak heights after complete alkaline hydrolysis of the lactones in 0.02 M NaOH for 2 to 24 h at 25°C.

Recombinant PON1 Cysteine Mutant. The cysteine at residue 284 of human PON1 type Q was replaced with alanine (C284A) using site-directed mutagenesis techniques as described previously (Sorenson et al., 1995). Serum-free Ultraculture media (Bio-Whittaker, Walkersville, MD) from Chinese hamster ovary cells stably expressing the C284A and wild-type (PON1 type Q) recombinants and a control cell line stably transfected with the pGS vector were concentrated 20-fold using Centricon-30 concentrators (Amicon, Beverly, MA) and assayed with phenyl acetate, paraoxon, the aromatic substrate homogentisic acid lactone, and the aliphatic substrate undecanoic--lactone.

Removal of Calcium from PON1 and Replacement with Metal Ions. A chelating resin (Chelex 100, Bio-Rad, Hercules, CA) was used to remove calcium from PON1 type Q enzyme as described (Kuo and La Du, 1998). Equal volumes of buffer containing either 2 mM calcium, magnesium, or zinc chloride were added to aliquots of enzyme preparation immediately after elution from the Chelex column. Care was taken to not irreversibly inactivate the enzyme after removal of the high-affinity bound calcium. Assays were performed with the appropriate metal present at 1 mM in the reaction cuvette.

Stimulation of PON1 Enzymatic Activities by Dilauroyl Phosphatidylcholine (PC). Two milligrams of dilauroyl PC were dissolved in 1.0 ml of 50 mM Tris/HCl (pH 8.0), 1.0 mM CaCl₂ buffer and dispersed by sonication for 45 sec. One volume of purified PON1 type Q or type R was mixed with an equal volume of the phospholipid suspension. Activities of stimulated and nonstimulated PON1 samples were obtained using phenyl acetate and the lactone substrates dihydrocoumarin, 2-coumaranone, homogentisic acid lactone, -butyrolactone, -caprolactone, and undecanoic--lactone. The percent stimulation or inhibition of PON1 with these substrates was estimated from changes in substrate hydrolysis after treatment.

PON1 Inhibition with Lactams. Inhibition of PON1 arylesterase activity by lactams was achieved through either addition of the lactam to the reaction cuvette (initial inhibition) or preincubation with the enzyme and reaction buffer for 15 min, followed by addition of the substrate (phenyl acetate) to initiate the reaction. IC₅₀ values were approximated by titrating arylesterase inhibition with the lactams. Reversibility was determined by incubating the enzyme with lactam for 1 h and then removing the lactam by washing with 4 volumes of enzyme buffer using a Centricon-30.

Determination of Kinetic Constants. Kinetics values were derived both from double reciprocal plots and by fitting experimental data to the Michaelis-Menten equation using GraphPad Prism software (version 3.00).

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

Previous investigations on PON1's hydrolytic activities with aromatic esters and organophosphates have identified a number of important properties that should be studied for a clear understanding of the enzyme's hydrolytic characteristics. Here we analyze PON1's lactonase activity using several of the same enzymatic characteristicsincluding substrate structural requirements, basic kinetics, important amino acids, and calcium requirementspreviously studied for its arylesterase and organophosphatase activities.

Substrate Specificity.

Lactone Substrates Lactones with varying ring sizes and substituents were tested with purified human PON1 types Q and R to gain information about the enzyme's structure/activity profile. To obtain values appropriate for comparison of the PON1 isozymes, we chose to relate all lactonase activities to the arylesterase activity with phenyl acetate. This reference compound was selected because the Q and R isoforms have approximately the same specific activity with this substrate under the above assay conditions. Purified PON1 Q and R preparations were diluted to have 100 units/ml arylesterase activity (with 1 mM substrate) for all subsequent assays unless otherwise specified. Current studies at this laboratory suggest that a suitable lactone may need to be selected for similar activity comparisons with other PON family members (i.e., PON2 and PON3) because of their limited or absent arylesterase activity.

Some Lactone Drugs Are Substrates for PON1. Based on the structural requirements for PON1's lactonase activity, it is reasonable to expect that some lactone- and carbonate ester-containing drugs or prodrugs should be hydrolyzed by this enzyme. We found that the diuretic spironolactone and three hydroxymethylglutaryl-CoA reductase inhibitors, i.e., mevastatin, lovastatin, and simvastatin, are hydrolyzed by PON1 (Table 4). Both Q and R isozymes of PON1 appear to hydrolyze these compounds with approximately the same efficiency under the experimental conditions used.

Thiolactones are less well hydrolyzed by PON1 than lactones. Thiolactones are hydrolyzed by PON1, but at much lower rate than the structurally homologous lactones (Table 1). Although HTL is a relatively poor substrate for PON1, it appears to be hydrolyzed only by PON1 in human serum (Jakubowski, 2000).

Lactams Inhibit PON1 Activity. Lactams are isosteric forms of lactones in which the ring oxygen is replaced by a nitrogen. It appears that lactams are not hydrolyzed by PON1 but rather inhibit the enzyme. The in vitro inhibition studies thus far performed have identified seven lactams as potent PON1 inhibitors: oxindole, isatin, -valerolactam, -caprolactam, 2-hydroxyquinoline, 3,4-dihydro-2(1H)-quinoline and N-bromo--caprolactam (Table 5). For some of these compounds (i.e., 2-hydroxyquinoline and 3,4-dihydro-2(1H)-quinoline), the IC₅₀ values are in the micromolar range. Isatin and N-bromo--caprolactam appear to inhibit PON1 arylesterase activity irreversibly; these lactams are being further investigated to identify residues that are presumably components of the enzyme's active center.

Enzyme Requirements for Lactonase Activity.

PON1's Free Cysteine (Residue 284) Is Important for Lactonase Activity Augustinsson and Ekedahl (1962) hypothesized that the mechanism of arylester hydrolysis involves the formation of thioester intermediates from the enzyme sulfhydryl and the aryl moiety of the substrate. We have previously demonstrated that PON1's free cysteine (residue 284) is not required for the arylesterase and paraoxonase activities (Sorenson et al., 1995), but is essential for the enzyme's ability to protect LDL against oxidation (Aviram et al., 1998). To determine whether the conserved free cysteine is required for PON1 lactonase activity, we compared wild-type recombinant PON1 type Q with a C284A mutant (Table 6).

Metal Ion Requirements. Both arylesterase and paraoxonase activities of PON1 require calcium for activity (Kuo and La Du, 1998). Activities of Ca²⁺ ion-depleted preparations in which Ca²⁺ ions were replaced by either Zn²⁺ or Mg²⁺ are compared with the calcium preparations in Table 7.

Modification of Lactonase Activity by Phospholipids: Stimulation by Dilauroyl PC. The paraoxonase and arylesterase activities of purified human serum PON1 are stimulated by a number of phospholipids with the greatest stimulation obtained in the presence of dilauroyl PC (Kuo and La Du, 1995). Stimulation is achieved through improvement of the enzyme's V_max but not K_m values. An interaction between serum PON1 and phospholipids is probably important due to the enzyme's strong association with lipids in HDL (Sorenson et al., 1999). Because of this association, we have tested stimulation of PON1 types Q and R with dilauroyl PC to determine whether it has an effect on lactonase activity as well.

Lactonase Evolution: Evolutionary Aspects of the PONs. There is appreciable structural homology between a fungal lactonase of Fusarium oxysporum and human PON1 (Kobayashi et al., 1998). Kobayashi et al. logically suggest an evolutionary relationship between these fungal and human enzymes, and recent work in our laboratory supports this idea. Surprisingly, compounds such as homogentisic acid lactone and dihydrocoumarin were substrates for the fungal lactonase (Kobayashi et al., 1998) and for both the human (this study) and rabbit (Watson et al., 2000) PON1 enzymes. The appreciable degree of sequence identity and homology between the other human PON-like proteins make it reasonable to expect that they also may have lactonase activity. Although this conservation of enzymatic activity does not appear true for arylesterase and organophosphatase activities, these two activities may reflect recently acquired characteristics as opposed to a more basic lactonase function. In fact, lactonase activity has recently been observed with rabbit PON3 (Draganov et al., 2000) and lactone substrates may be helpful in identifying other members of the mammalian PON family.