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• W2085382883 abstract "Serum paraoxonases (PONs) are calcium-dependent lactonases that catalyze the hydrolysis and formation of a variety of lactones, with a clear preference for lipophilic lactones. However, the lactonase mechanism of mammalian PON1, a high density lipoprotein-associated enzyme that is the most studied family member, remains unclear, and other family members have not been examined at all. We present a kinetic and site-directed mutagenesis study aimed at deciphering the lactonase mechanism of PON1 and PON3. The pH-rate profile determined for the lactonase activity of PON1 indicated an apparent pKa of ∼7.4. We thus explored the role of all amino acids in the PON1 active site that are not directly ligated to the catalytic calcium and that possess an imidazolyl or carboxyl side chain (His115, His134, His184, His285, Asp183, and Asp269). Extensive site-directed mutagenesis studies in which each amino acid candidate was replaced with all other 19 amino acids were conducted to identify the residue(s) that mediate the lactonase activity of PONs. The results indicate that the lactonase activity of PON1 and PON3 and the esterase activity of PON1 are mediated by the His115-His134 dyad. Notably, the phosphotriesterase activity of PON1, which is a promiscuous activity of this enzyme, is mediated by other residues. To our knowledge, this is one of few examples of a histidine dyad in enzyme active sites and the first example of a hydrolytic enzyme with such a dyad. Serum paraoxonases (PONs) are calcium-dependent lactonases that catalyze the hydrolysis and formation of a variety of lactones, with a clear preference for lipophilic lactones. However, the lactonase mechanism of mammalian PON1, a high density lipoprotein-associated enzyme that is the most studied family member, remains unclear, and other family members have not been examined at all. We present a kinetic and site-directed mutagenesis study aimed at deciphering the lactonase mechanism of PON1 and PON3. The pH-rate profile determined for the lactonase activity of PON1 indicated an apparent pKa of ∼7.4. We thus explored the role of all amino acids in the PON1 active site that are not directly ligated to the catalytic calcium and that possess an imidazolyl or carboxyl side chain (His115, His134, His184, His285, Asp183, and Asp269). Extensive site-directed mutagenesis studies in which each amino acid candidate was replaced with all other 19 amino acids were conducted to identify the residue(s) that mediate the lactonase activity of PONs. The results indicate that the lactonase activity of PON1 and PON3 and the esterase activity of PON1 are mediated by the His115-His134 dyad. Notably, the phosphotriesterase activity of PON1, which is a promiscuous activity of this enzyme, is mediated by other residues. To our knowledge, this is one of few examples of a histidine dyad in enzyme active sites and the first example of a hydrolytic enzyme with such a dyad. Serum paraoxonases (PONs) 2The abbreviations used are: PONs, serum paraoxonases; rePON1, recombinant PON1; TBBL, 5-(thiobutyl)butyrolactone; MES, 4-morpholineethanesulfonic acid; bis-tris propane, 1,3-bis(tris(hydroxymethyl)methylamino)propane.2The abbreviations used are: PONs, serum paraoxonases; rePON1, recombinant PON1; TBBL, 5-(thiobutyl)butyrolactone; MES, 4-morpholineethanesulfonic acid; bis-tris propane, 1,3-bis(tris(hydroxymethyl)methylamino)propane. constitute a family of calcium-dependent mammalian enzymes that have been recently defined as lipophilic lactonases. PON1 is the best studied member of the family, with other members being PON2 and PON3 (1Draganov D.I. La Du B.N. Naunyn-Schmiedeberg's Arch. Pharmacol. 2004; 369: 78-88Crossref PubMed Scopus (359) Google Scholar, 2La Du B.N. Aviram M. Billecke S. Navab M. Primo-Parmo S. Sorenson R.C. Standiford T.J. Chem. Biol. Interact. 1999; 119-120: 379-388Crossref PubMed Scopus (172) Google Scholar). PON1 catalyzes the hydrolysis of multiple substrates: lactones, thiolactones, carbonates, esters, and phosphotriesters, including paraoxon, from which its name is derived. However, only after a few decades of research, it became apparent that PON1 and the other PONs are in fact lactonases (3Aharoni A. Gaidukov L. Khersonsky O. Gould S.M. Roodveldt C. Tawfik D.S. Nat. Genet. 2005; 37: 73-76Crossref PubMed Scopus (643) Google Scholar, 4Draganov D.I. Teiber J.F. Speelman A. Osawa Y. Sunahara R. La Du B.N. J. Lipid Res. 2005; 46: 1239-1247Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar, 5Gaidukov L. Tawfik D.S. Biochemistry. 2005; 44: 11843-11854Crossref PubMed Scopus (184) Google Scholar, 6Khersonsky O. Tawfik D.S. Biochemistry. 2005; 44: 6371-6382Crossref PubMed Scopus (360) Google Scholar), catalyzing both the hydrolysis (4Draganov D.I. Teiber J.F. Speelman A. Osawa Y. Sunahara R. La Du B.N. J. Lipid Res. 2005; 46: 1239-1247Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar, 6Khersonsky O. Tawfik D.S. Biochemistry. 2005; 44: 6371-6382Crossref PubMed Scopus (360) Google Scholar) and formation (7Teiber J.F. Draganov D.I. La Du B.N. Biochem. Pharmacol. 2003; 66: 887-896Crossref PubMed Scopus (129) Google Scholar) of a variety of lactones. Structure-reactivity studies (6Khersonsky O. Tawfik D.S. Biochemistry. 2005; 44: 6371-6382Crossref PubMed Scopus (360) Google Scholar) and laboratory evolution experiments (3Aharoni A. Gaidukov L. Khersonsky O. Gould S.M. Roodveldt C. Tawfik D.S. Nat. Genet. 2005; 37: 73-76Crossref PubMed Scopus (643) Google Scholar) indicate that the native activity of PON1 is lactonase. The other activities, e.g. arylesterase and phosphotriesterase (paraoxonase), are merely promiscuous and are not shared by other family members, e.g. PON2 and PON3. PON1 activation by binding to high density lipoprotein particles carrying apoA-I also indicates high specificity toward lactone substrates and, in particular, lipophilic lactones that display kcat/Km values of 106-107 m-1 s-1 (5Gaidukov L. Tawfik D.S. Biochemistry. 2005; 44: 11843-11854Crossref PubMed Scopus (184) Google Scholar). The physiological substrates of PONs are still unknown, but they are likely to include lactones consumed as food ingredients (8Adams T.B. Greer D.B. Doull J. Munro I.C. Newberne P. Portoghese P.S. Smith R.L. Wagner B.M. Weil C.S. Woods L.A. Ford R.A. Food Chem. Toxicol. 1998; 36: 249-278Crossref PubMed Scopus (75) Google Scholar) or derivatives of fatty acid oxidation processes, e.g. 5-hydroxyeicosatetraenoic acid lactone (4Draganov D.I. Teiber J.F. Speelman A. Osawa Y. Sunahara R. La Du B.N. J. Lipid Res. 2005; 46: 1239-1247Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar, 8Adams T.B. Greer D.B. Doull J. Munro I.C. Newberne P. Portoghese P.S. Smith R.L. Wagner B.M. Weil C.S. Woods L.A. Ford R.A. Food Chem. Toxicol. 1998; 36: 249-278Crossref PubMed Scopus (75) Google Scholar), that reside in high density lipoprotein, low density lipoprotein, or macrophage cells. PON1 is composed of 354 amino acids. The enzyme has two calcium-binding sites: the higher affinity calcium is required for the structural integrity, whereas the lower affinity calcium is involved in catalysis (9Kuo C.L. La Du B.N. Drug Metab. Dispos. 1998; 26: 653-660PubMed Google Scholar). Early mechanistic studies using chemical labeling and site-directed mutagenesis identified several residues that are involved in the phosphotriesterase and esterase activities of human PON1 (10Josse D. Xie W. Renault F. Rochu D. Schopfer L.M. Masson P. Lockridge O. Biochemistry. 1999; 38: 2816-2825Crossref PubMed Scopus (86) Google Scholar, 11Josse D. Lockridge O. Xie W. Bartels C.F. Schopfer L.M. Masson P. J. Appl. Toxicol. 2001; 21: S7-S11Crossref PubMed Scopus (74) Google Scholar). However, because these studies were conducted before the three-dimensional structure of PON1 was known, it was largely unclear whether these amino acids are indeed in the PON1 active site or whether they are involved in substrate binding, Ca2+ binding, or catalysis. Recently, a crystal structure of a recombinant PON1 (rePON1) variant (G2E6) was solved at a resolution of 2.2Å, providing the first structure of a PON family member (12Harel M. Aharoni A. Gaidukov L. Brumshtein B. Khersonsky O. Meged R. Dvir H. Ravelli R.B. McCarthy A. Toker L. Silman I. Sussman J.L. Tawfik D.S. Nat. Struct. Mol. Biol. 2004; 11: 412-419Crossref PubMed Scopus (530) Google Scholar). This variant was directly evolved from rabbit PON1. It is expressed in a soluble and active form in Escherichia coli and exhibits enzymatic properties that are essentially identical to those reported for PON1 purified from sera. PON1 was found to be a six-bladed β-propeller, with the two calcium ions located in the central tunnel. The structural calcium (Ca2) is buried, whereas the catalytic calcium (Ca1) is solvent-exposed and located at the bottom of a deep hydrophobic active site. The active-site residues of PON1 were also defined by amino acids whose alteration during directed evolution shifted the activity and substrate selectivity of PON1. The structure of PON1 allowed us to postulate its mechanism of catalysis (12Harel M. Aharoni A. Gaidukov L. Brumshtein B. Khersonsky O. Meged R. Dvir H. Ravelli R.B. McCarthy A. Toker L. Silman I. Sussman J.L. Tawfik D.S. Nat. Struct. Mol. Biol. 2004; 11: 412-419Crossref PubMed Scopus (530) Google Scholar). On the basis of pH-rate profiles constructed for paraoxon and 2-naphthyl acetate hydrolysis, an unprotonated histidine was supposed to be involved in the base-catalyzed rate-determining step of catalysis by PON1. A histidine dyad composed of His115 and His134 was suggested to be directly involved in the catalytic mechanism of PON1 for both ester and phosphotriester hydrolysis. Mutagenesis experiments supported the suggested mechanism, although it was later found that these mutants were probably misfolded and therefore inactive (13Harel M. Aharoni A. Gaidukov L. Brumshtein B. Khersonsky O. Meged R. Dvir H. Ravelli R.B. McCarthy A. Toker L. Silman I. Sussman J.L. Tawfik D.S. Nat. Struct. Mol. Biol. 2004; 11: 1253Crossref Scopus (9) Google Scholar). Moreover, Yeung et al. (14Yeung D.T. Josse D. Nicholson J.D. Khanal A. McAndrew C.W. Bahnson B.J. Lenz D.E. Cerasoli D.M. Biochim. Biophys. Acta. 2004; 1702: 67-77Crossref PubMed Scopus (66) Google Scholar) recently reported that the H115W mutant of human PON1 retains activity with paraoxon. They therefore postulated that His115 is important for substrate binding and specificity, but does not directly participate in catalysis (15Yeung D.T. Lenz D.E. Cerasoli D.M. FEBS J. 2005; 272: 2225-2230Crossref PubMed Scopus (51) Google Scholar). Most important, all previous mechanistic studies of PON1 addressed the phosphotriesterase and esterase activities, but the mechanism of lactone hydrolysis, which now appears to be the primary function of PON1, was not explored. The mechanism of other mammalian PON family members, most notably PON3, which exhibits weak esterase activity and almost no paraoxonase activity, has not been studied either. This study aimed to decipher the lactonase mechanism of PON1 and PON3. We determined the pH-rate profile for lactone hydrolysis and conducted extensive site-directed mutagenesis studies to identify the residues that mediate this activity. We show that the lactonase and esterase activities of PON1 are mediated by the His115-His134 dyad and rule out other active-site residues, including His285. Finally, the accompanying article (16Rosenblat M. Gaidukov L. Khersonsky O. Vaya J. Oren R. Tawfik D.S. Aviram M. J. Biol. Chem. 2006; 281: 7657-7665Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) shows that the PON1 mutants with reduced lactonase activity studied here (H115Q, H134Q, and the double mutant H115Q/H134Q) exhibit reduced or no biological function in ex vivo assay. We also show that the paraoxonase activity is promiscuous in terms of substrate binding, but is also mediated by residues other than those that mediate the native activity. Materials—Chemicals were purchased from Aldrich, Fluka, and Acros Organics. Primers for site-directed mutagenesis were purchased from Sigma. Site-directed Mutagenesis—The pET32b(+) plasmid containing the gene for rePON1-G2E6 (12Harel M. Aharoni A. Gaidukov L. Brumshtein B. Khersonsky O. Meged R. Dvir H. Ravelli R.B. McCarthy A. Toker L. Silman I. Sussman J.L. Tawfik D.S. Nat. Struct. Mol. Biol. 2004; 11: 412-419Crossref PubMed Scopus (530) Google Scholar) was used as a template for PCR amplification. The mutants were constructed by the “inverse PCR” method using two neighboring non-overlapping primers, one of which bears the mutation at its 5′-end (17Ling M.M. Robinson B.H. Anal. Biochem. 1997; 254: 157-178Crossref PubMed Scopus (183) Google Scholar). Pfu Turbo polymerase was applied for 25 cycles of polymerization at 72 °C. After digestion of the template plasmid with DpnI, the amplified DNA was blunt-ligated with T4 ligase, and the ligated DNA was transformed into E. coli DH5α cells. The mutated genes were verified by DNA sequencing. The histidine replacement libraries were generated by replacing His codons with DNS codons (where D is an equimolar mixture of A, G, and T; N is a mixture of all four nucleotides; and S is a mixture of C and G) encoding all amino acids except His, Gln, and Pro. The Gln replacement libraries were generated separately by replacing the His codon with a Gln codon (CAG). The aspartate replacement libraries were produced by introducing a combination of NNR and HNS degeneracy codons (where R is an equimolar mixture of A and G, and H is an equimolar mixture of A, C, and T) encoding all amino acids except Asp. Screening of rePON1 Mutants—The libraries were transformed into E. coli Origami B DE3 cells (Novagen). Colonies grown on agar were used to inoculate 500 μl of LB medium in a 96-well plate and were grown overnight at 37 °C. The plates were duplicated; lysed with Bug-Buster (Novagen); and screened for esterase (2-naphthyl acetate; 0.2 mm), phosphotriesterase (paraoxon; 0.5 mm), and lactonase (5-(thiobutyl)butyrolactone (TBBL) (18Khersonsky O. Tawfik D. ChemBioChem. 2005; 7: 49-53Crossref Scopus (77) Google Scholar); 0.22 mm) activities. Expression and Purification of rePON1-G2E6 Mutants—Wild type-like rePON1-G2E6 and its various mutants were expressed as fusion proteins with thioredoxin and His6 tags and purified as described previously (19Aharoni A. Gaidukov L. Yagur S. Toker L. Silman I. Tawfik D.S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 482-487Crossref PubMed Scopus (256) Google Scholar), except that mutant proteins were eluted from a nickel-nitrilotriacetic acid column in buffer containing 10% glycerol, 0.1% Tergitol, and 50 mm NaCl. The purity of wild-type rePON1 and its mutants was analyzed by SDS-PAGE, and the proteins were essentially pure (>90%). The expression levels of wild-type rePON1 and the purified mutants were 20-50 mg/liter of culture. pH-rate Profile—kcat and Km values were determined for rePON1-G2E6 with TBBL (18Khersonsky O. Tawfik D. ChemBioChem. 2005; 7: 49-53Crossref Scopus (77) Google Scholar) at pH 5.8-9.4. Initial velocities (v0) were determined at eight different concentrations for each substrate. The buffers used were MES (pH 5.8-6.5) and bis-tris propane (pH 6.5-9.4) at 0.1 m plus 1 mm CaCl2. The ionic strength was adjusted to a total of 0.2 m with NaCl. The enzyme stocks were kept in 50 mm Tris containing 0.1% Tergitol, 50 mm NaCl, and 1 mm CaCl2. TBBL (18Khersonsky O. Tawfik D. ChemBioChem. 2005; 7: 49-53Crossref Scopus (77) Google Scholar) was used from a 0.2 m stock in acetonitrile, and the co-solvent percentage was equalized to 1% in all reaction mixtures. Product formation was monitored spectro-photometrically in 200-μl reaction volumes using 96-well plates by coupling to 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman's reagent) as described (18Khersonsky O. Tawfik D. ChemBioChem. 2005; 7: 49-53Crossref Scopus (77) Google Scholar). For assays at pH ≤7.0 (below the pKa of 5,5′-dithiobis(2-nitrobenzoic acid)), 100-μl aliquots taken from 1-ml reactions were transferred at 20-s intervals into 100 μl of buffer containing 5,5′-dithiobis(2-nitrobenzoic acid) and the PON1 inhibitor 2-hydroxyquinoline (2 mm) to quench the enzymatic reaction. The product concentration was subsequently determined by absorbance at 412 nm. Initial velocities were determined by plotting these end point measurements against time (five or more points) and extrapolating a slope for the linear phase. The reported results are the average of at least three independent measurements. Kinetic Measurements with rePON1 Mutants—The kinetic measurements were performed in buffer containing 50 mm Tris (pH 8.0) and 1 mm CaCl2, and aliphatic lactone hydrolysis was monitored as described previously (6Khersonsky O. Tawfik D.S. Biochemistry. 2005; 44: 6371-6382Crossref PubMed Scopus (360) Google Scholar). A range of enzyme concentrations was used depending on the reactivity of the substrate and the mutant. The activities of the mutants and wild type-like rePON1-G2E6 were examined with substrates of PON1 of three subgroups: phosphotriesters (paraoxon, 0.85 mm; and 7-diethylphosphoro-3-cyanocoumarin (DEP-coumarin, 47.5 μm), esters (phenyl acetate, 1 mm; and 2-naphthyl acetate, 0.2 mm), and lactones (dihydrocoumarin, 0.25 mm; δ-valerolactone, 1 mm; and γ-nonalactone, 1 mm). The substrate concentrations were varied according to the solubility, reactivity, and extinction coefficients of each substrate. The reported results are the average of at least two independent measurements. For kinetic parameters determinations, the substrate concentrations were varied in the range of 0.3 × Km up to 2-3 × Km, except for those cases in which substrate solubility was limiting (phenyl acetate in the case of all mutants, δ-valerolactone in the case of H115Q and the double mutant, and γ-caprolactone in the case of H115Q and H134Q). The percentage of co-solvent (MeOH in case of phenyl acetate and paraoxon, Me2SO in case of lactones, and acetonitrile in the case of TBBL) was equalized to 1-1.6% in all reactions. Data Analysis—Kinetic parameters (kcat, Km, and kcat/Km) were obtained by fitting the data to the Michaelis-Menten equation (v0 = kcat[E]0[S]0/([S]0 + Km)) using the program KaleidaGraph 5.0. In cases in which solubility limited the initial substrate concentrations, the data were fitted to the linear regime of the Michaelis-Menten model (v0 = [S]0[E]0kcat/Km), and kcat/Km was deduced from the slope. All data presented are the means ± S.D. of at least three independent experiments. The pH-rate data (kcat ((kcat)H) and kcat/Km ((kcat/Km)H) values for each pH value) were fitted to a “bell-shaped” model using the equations (kcat)H = (kcat)max/((10-pH/10-pKa1) + (10-pKa2/10-pH) + 1) and (k/Km)H = (kcat/Km)max/((10-pH/10-pKa1) + (10-pKa2/10-pH) + 1), where (kcat)max and (kcat/Km)max are the plateau values of kcat and kcat/Km, respectively, and pKa1 and pKa2 are the apparent pKa values for the acidic and basic groups, respectively. Rabbit PON3—Wild-type rabbit PON3 and its mutants were cloned, expressed, and purified analogously to rePON1 variants, except that the elution buffer did not contain glycerol. The expression of PON3 variants was comparatively low (∼2 mg/liter of culture) and was entirely dependent on the fusion protein thioredoxin (19Aharoni A. Gaidukov L. Yagur S. Toker L. Silman I. Tawfik D.S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 482-487Crossref PubMed Scopus (256) Google Scholar). Although SDS-PAGE indicated that the resulting protein was only 30% pure, the kinetic analysis was performed without any further purification because no contaminating lactonase activity was observed. The ratios between the activities of PON1 and PON3 with several aliphatic lactones were similar to those reported by Draganov et al. (4Draganov D.I. Teiber J.F. Speelman A. Osawa Y. Sunahara R. La Du B.N. J. Lipid Res. 2005; 46: 1239-1247Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar) and Billecke et al. (20Billecke S. Draganov D. Counsell R. Stetson P. Watson C. Hsu C. La Du B.N. Drug Metab. Dispos. 2000; 28: 1335-1342PubMed Google Scholar). However, the esterase activities of rabbit PON3 with phenyl acetate and p-nitrophenyl acetate were found to be much lower than reported previously (4Draganov D.I. Teiber J.F. Speelman A. Osawa Y. Sunahara R. La Du B.N. J. Lipid Res. 2005; 46: 1239-1247Abstract Full Text Full Text PDF PubMed Scopus (551) Google Scholar). The activities of PON3 and its mutants were determined with 5-(thioethyl)butyrolactone (TEBL, 0.22 mm), TBBL (0.27 mm), δ-valerolactone (1 mm), γ-nonalactone (1 mm), and γ-undecanoic lactone (0.5 mm). The hydrolysis of aliphatic lactones is usually measured by the pH indicator assay (5Gaidukov L. Tawfik D.S. Biochemistry. 2005; 44: 11843-11854Crossref PubMed Scopus (184) Google Scholar, 6Khersonsky O. Tawfik D.S. Biochemistry. 2005; 44: 6371-6382Crossref PubMed Scopus (360) Google Scholar, 7Teiber J.F. Draganov D.I. La Du B.N. Biochem. Pharmacol. 2003; 66: 887-896Crossref PubMed Scopus (129) Google Scholar), which does not allow wide variations of pH. Thus, the pH-rate profile of rePON1 was determined with TBBL (18Khersonsky O. Tawfik D. ChemBioChem. 2005; 7: 49-53Crossref Scopus (77) Google Scholar), a lactone substrate that is analogous to γ-nonanoic lactone, yet releases, upon hydrolysis of the γ-butyrolactone ring, a thiol moiety that can be detected with Ellman's reagent (5,5′-dithiobis(2-nitrobenzoic acid)). An overlay of the pH-rate profile of TBBL with the previously published pH-rate profiles of an ester (2-naphthyl acetate) and a phosphotriester (paraoxon) (12Harel M. Aharoni A. Gaidukov L. Brumshtein B. Khersonsky O. Meged R. Dvir H. Ravelli R.B. McCarthy A. Toker L. Silman I. Sussman J.L. Tawfik D.S. Nat. Struct. Mol. Biol. 2004; 11: 412-419Crossref PubMed Scopus (530) Google Scholar) is provided in Fig. 1. The parameters obtained from the pH-rate profiles are summarized in Table 1.TABLE 1Kinetic parameters extracted from the pH-rate profiles of rePON1Substrate(kcat)maxpKa1 (kcat data)pKa2 (kcat data)(kcat/Km)maxpKa1 (kcat/Km data)pKa2 (kcat/Km data)S−1M−1 S−1TBBL104 ± 117.4 ± 0.1~9.8apKa2 values were extrapolated from the data obtained below the actual pKa2 and hence can only be estimated.750,000 ± 150,0007.2 ± 0.28.7 ± 0.22-Naphthyl acetate18 ± 16.67 ± 0.098.9 ± 0.1170,000 ± 17,6007.2 ± 0.19.0 ± 0.1Paraoxon1.09 ± 0.086.3 ± 0.1~10.0apKa2 values were extrapolated from the data obtained below the actual pKa2 and hence can only be estimated.6980 ± 2907.04 ± 0.06~9.8apKa2 values were extrapolated from the data obtained below the actual pKa2 and hence can only be estimated.a pKa2 values were extrapolated from the data obtained below the actual pKa2 and hence can only be estimated. Open table in a new tab Overall, the pH-rate profiles of all three substrates are similar, with a fully pronounced acidic shoulder and a minor basic shoulder that is most apparent in the case of 2-naphthyl acetate. With the exception of TBBL, for which Km values obtained at pH <6.5 were higher than those obtained at pH >6.5, the Km values did not vary much with pH, as indicated by the similarity of the pH-rate profiles obtained for kcat and kcat/Km. The difference between the pKa values obtained for kcat versus kcat/Km can be explained by the fact that the kcat/Km data provide the pKa of the free enzyme, whereas the kcat data provide the pKa of the enzyme-substrate complex. The small differences between the pKa values obtained with same substrate for kcat versus kcat/Km may therefore reflect a change in the active-site environment upon formation of the enzyme-substrate complex. Notably, the pKa1 derived from kcat/Km is essentially identical for all substrates, thus reflecting the same free enzyme form. The larger differences observed with the pKa2 derived from kcat/Km, especially for paraoxon, may indicate that this substrate might be binding a different conformer of PON1. It should also be noted that pKa2 values were largely extrapolated from the data obtained around or even below pKa2; thus, the accuracy of pKa2 values is inevitably low. As in the case of 2-naphthyl acetate and paraoxon (12Harel M. Aharoni A. Gaidukov L. Brumshtein B. Khersonsky O. Meged R. Dvir H. Ravelli R.B. McCarthy A. Toker L. Silman I. Sussman J.L. Tawfik D.S. Nat. Struct. Mol. Biol. 2004; 11: 412-419Crossref PubMed Scopus (530) Google Scholar), the major acidic shoulder (pKa1) of the lactone substrate may be ascribed to a group that directly participates in catalysis and is active in its basic deprotonated form. The observed pKa values (6.3-7.4) are most consistent with the imidazole group of histidine, the pKa of which in aqueous solution is ∼6.8 (21Copeland R.A. Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis. 2nd. Wiley-VCH, New York2000Crossref Google Scholar). However, because the pKa values of side chains in proteins and particularly in active sites can vary greatly from their values in solution, other residues could not be excluded on the basis of the pH-rate profiles. The observed pKa can also correspond to an aspartic or glutamic acid side chain, the pKa of which is generally ∼4, but can be raised in enzyme active sites up to and possibly beyond 6.5 (21Copeland R.A. Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis. 2nd. Wiley-VCH, New York2000Crossref Google Scholar). The differences in the acidic pKa values of the different substrates, specifically those obtained with kcat, may reflect variations in the catalytic mechanism. Most notable is the difference between the pKa1 of paraoxon (6.3) and that of TBBL (7.4). Indeed, as shown below, the hydrolysis of paraoxon is not mediated by the same active-site residues that mediate the lactonase activity. The minor basic shoulder (pKa2) probably reflects a deprotonation of a basic side chain that affects the active site, but is not directly involved in catalysis. Among other possibilities, this basic pKa might reflect a general deprotonation of lysine side chains (e.g. Lys192) that causes a mild deactivation of the enzyme. Although at this stage we cannot completely rule out a nucleophilic mechanism, we have not observed any kinetic indications for the existence of an acyl-enzyme intermediate, not even in substrates with very good leaving groups (6Khersonsky O. Tawfik D.S. Biochemistry. 2005; 44: 6371-6382Crossref PubMed Scopus (360) Google Scholar). Thus, we assume the simplest mechanism in which an active-site general base deprotonates a water molecule to generate a hydroxide ion that attacks the phosphoryl/carbonyl of the various substrates. The active site of PON1 (Fig. 2) contains not one but several reasonable candidates for the role of a general base, including four histidines (His115, His134, His285, and His184), two aspartates (Asp183 and Asp269), and one glutamic acid (Glu53). All these residues are conserved in all mammalian PONs. Asp269 and Glu53 participate in the ligation of catalytic Ca2+; and although they are not likely to act as a general base (let alone as a nucleophile), their titration could, in principle, disrupt the active site and give rise to pKa1. Thus, a mechanism in which Asp269 or Glu53 is involved in both Ca2+ ligation and water deprotonation cannot be totally ruled out at this stage. His115, being only ∼4 Å from the catalytic calcium, is the most obvious candidate for the general base role. As proposed previously (12Harel M. Aharoni A. Gaidukov L. Brumshtein B. Khersonsky O. Meged R. Dvir H. Ravelli R.B. McCarthy A. Toker L. Silman I. Sussman J.L. Tawfik D.S. Nat. Struct. Mol. Biol. 2004; 11: 412-419Crossref PubMed Scopus (530) Google Scholar), it can form a His-His dyad with His134, in which His115 is activated by His134 via a proton shuttle mechanism. His134 itself is far less likely to act directly as a general base because of its distance from the catalytic calcium (8.7 Å). His285 is also not far from the catalytic calcium (∼7 Å) and was indeed suggested to be involved in catalysis (14Yeung D.T. Josse D. Nicholson J.D. Khanal A. McAndrew C.W. Bahnson B.J. Lenz D.E. Cerasoli D.M. Biochim. Biophys. Acta. 2004; 1702: 67-77Crossref PubMed Scopus (66) Google Scholar). Another possible candidate for the general base role is His184, although its distance from the catalytic calcium is quite long (∼11 Å). Asp183, which neighbors His184, could either act itself as a general base or form a dyad with His184, in which His184 acts as a general base and Asp183 as a proton shuttle. Similar arrangements are observed in other calcium-dependent hydrolases such as secreted phospholipase A2 and diisopropyl-fluorophosphatase (22Scharff E.I. Koepke J. Fritzsch G. Lucke C. Ruterjans H. Structure (Camb). 2001; 9: 493-502Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 23Sekar K. Yu B.Z. Rogers J. Lutton J. Liu X. Chen X. Tsai M.D. Jain M.K. Sundaralingam M. Biochemistry. 1997; 36: 3104-3114Crossref PubMed Scopus (51) Google Scholar). Given this variety of putative catalytic residues, we applied site-directed mutagenesis to identify those side chains that mediate the lactonase activity of mammalian PONs and possibly the other promiscuous activities of PON1. Although an extremely powerful tool, site-directed mutagenesis has its own limitations (24Peracchi A. Trends Biochem. Sci. 2001; 26: 497-503Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 25Plapp B.V. Methods Enzymol. 1995; 249: 91-119Crossref PubMed Scopus (58) Google Scholar). Mutagenesis in the context of mechanistic studies generally involves substitutions of side chains with side chains that are similar in terms of size and polarity but that are unable to perform the same catalytic function, thu" @default.
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