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PubMed1997

ADP-ribosylarginine hydrolases and ADP-ribosyltransferases. Partners in ADP-ribosylation cycles.

Mono-ADP-ribosylation is a reversible modification of arginine residues in proteins, with NAD:arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases constituting opposing arms of a putative ADP-ribosylation cycle. The enzymatic components of an ADP-ribosylation cycle have been identified in both prokaryotic and eukaryotic systems. The regulatory significance of the cycle has been best documented in prokaryotes. As shown by Ludden and coworkers, ADP-ribosylation controls the activity of dinitrogenase reductase in the phototropic bacterium Rhodospirillum rubrum. ADP-ribosylation of other amino acids, such as cysteine, has also been demonstrated, lending credence to the hypothesis that this modification is heterogeneous. In eukaryotes, the functional relationship between ADP-ribosyltransferases and ADP-ribosylarginine hydrolases is less well documented. The transferase-catalyzed reaction results in sterospecific formation of alpha-ADP-ribosylarginine from beta-NAD; ADP-ribosylarginine hydrolases specifically cleave the alpha-anomer, leading to release of ADP-ribose and regeneration of the free guanidino group of arginine. The two reactions can thus be coupled in vitro. Coupling in vivo is dependent on cellular localization. The deduced amino acid sequences of ADP-ribosyltransferases from avian and mammalian tissues have common consensus sequences involved in catalytic activity but, in some instances, enzyme-specific cellular localization signals. The presence of amino- and carboxy-terminal signal sequences is consistent with the glycosylphosphatidylinositol(GPI)-anchoring to the cell surface. The muscle and lymphocyte transferases ADP-ribosylate integrins. Some transferases lack the carboxy- terminal signal sequence needed for GPI-anchoring. Most ADP-ribosylarginine hydrolase activity is cytosolic, although perhaps some is located at the cell surface. Deduced amino acid sequences of hydrolases from a number of mammalian species are consistent with their cytoplasmic localization. Katada and coworkers have determined, however, that auto-ADP-ribosylated RT6, a GPI-linked protein, is metabolized by a hydrolase-like activity, consistent with the existence of an ADP-ribosylation cycle. ADP-ribosyl RT6 may be internalized, thereby coming in contact with the cytosolic hydrolase; alternatively, a novel form of the hydrolase may be located at the surface. The mechanism of coupling of ADP-ribosyltransferases and hydrolases in eukaryotic ADP-ribosylation cycles has yet to be clarified.

ADP Ribose Transferases↗
PubMed1978

Extracellular hydrolases of the lung.

A pool of acid hydrolases exists within the acellular lining material of the alveoli and distal airways of the lungs. These extracellular hydrolases, obtained using pulmonary lavage procedures, appear to be of a selected variety insofar as some hydrolases (beta-N-acetylglucosaminidase and alpha-mannosidase) are highly active while others (beta-glucuronidase and arylsulfatase) are barely detectable. The origins of these hydrolases were investigated. Neither leakage of serum nor cell damage can account for the presence of the extracellular hydrolases in lavage effluents. Electrophoretic mobilities on acrylamide gels indicate that the extracellular hydrolases generally differ from those found in serum. Cytoplasmic soluble enzymes such as lactate dehydrogenase were used to monitor cell damage and show that the extracellular hydrolases did not originate from cell leakage during the lavage procedure. Hydrolases similar to those found extracellularly are associated with highly purified lysosome-free lamellar bodies isolated from homogenates of lung. The extracellular hydrolases are probably selected by the type 2 cells of the pulmonary alveolar epithelium during their selection of lamellar bodies.

Animals↗
PubMed1989

The presence of acyl-CoA hydrolase in rat brown-adipose-tissue peroxisomes.

The subcellular distribution of acyl-CoA hydrolase was studied in rat brown adipose tissue, with special emphasis on possible peroxisomal localization. Subcellular fractionation by sucrose-density-gradient centrifugation, followed by measurement of short-chain (propionyl-CoA) acyl-CoA hydrolase in the presence of NADH, resulted in two peaks of activity in the gradient: one peak corresponded to the distribution of cytochrome oxidase (mitochondrial marker enzyme), and another peak of activity coincided with the peroxisomal marker enzyme catalase. The distribution of the NADH-inhibited short-chain hydrolase activity fully resembled that of cytochrome oxidase. The substrate-specificity curve of the peroxisomal acyl-CoA hydrolase activity indicated the presence of a single enzyme exhibiting a broad substrate specificity, with maximal activity towards fatty acids with chain lengths of 3-12 carbon atoms. The mitochondrial acyl-CoA hydrolase substrate specificity, in contrast, indicated the presence of at least two acyl-CoA hydrolases (of short- and medium-chain-length specificity). The peroxisomal acyl-CoA hydrolase activity was inhibited by CoA at low (microM) concentrations and by ATP at high concentrations (greater than 0.8 mM). In contrast with the mitochondrial short-chain hydrolase, the peroxisomal acyl-CoA hydrolase activity was not inhibited by NADH.

Adipose Tissue, Brown↗
PubMed2000

Ecto-diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A)-hydrolase is expressed as an ectoenzyme in a variety of mammalian and human cells and adds new aspects to the turnover of Ap4A.

Ap4A and other dinucleotides participate in the regulation of hemostasis and blood pressure control. With the exception of two previously reported surface anchored ectoAp4A-hydrolases on bovine aortic endothelial and chromaffine cells, all Ap4A-hydrolases reported are intracellular or freely soluble. We demonstrated that ectoAp4A-hydrolases are present on a broad variety of cell types of different species: rat mesangial, bovine corneal epithelial, human Hep-G2 and peridontal cells. Ectoenzyme properties were evaluated on rat mesangium cells. Chromatography of purified plasma membranes on Sephacel 300 resulted in enrichment of ectoAp4A-hydrolase and in separation from ectoATPase. In contrast to ATPase, Ap4A-hydrolase was stable at room temperature. EctoAp4A-hydrolase also recognized ATP as substrate, and therefore is not highly specific. The molecular weight was 180 kD. Unlike ectoAMPase ectoAp4A-hydrolase was not attached via a glycosyl-phosphatidylinositol (GPI)-moiety. Concentrations of PI-PLC 10-100-fold higher than effective for ectoAMPase cleavage (10-100 mU/ml) plus extensively extended incubation times up to eight hours did not result in cleavage of ectoAp4A-hydrolase. The enzyme ectoAp4A-hydrolase might presage a direction for pharmaceutical manipulation in the control of blood pressure and hemostasis.

Acid Anhydride Hydrolases↗
PubMed2006

Novel conserved hydrolase domain in the CLCA family of alleged calcium-activated chloride channels.

Advanced protein structure prediction methods combined with structure modeling show that the mammalian proteins, described until now as calcium-activated chloride channels (CLCAs), appear in fact to be membrane anchored metal-dependent hydrolases, possibly proteases. A metallohydrolase structural domain was predicted, unexpectedly, in the CLCA sequences. The well-conserved active site in the modeled structure of this hydrolase domain allows the prediction of catalytic action similar to that of metalloproteases. A number of protein structure prediction methods suggest the overall fold of the N-terminal hydrolase domain to be most similar to that of zinc metalloproteases (zincins), notably matrixins. This is confirmed by analysis of the three-dimensional structure model of the predicted CLCA1 hydrolase domain built using the known structure of the MMP-11 catalytic domain. Fragments of CLCA1 corresponding to the modeled hydrolase domain were expressed in Escherichia coli, and the resulting proteins were readily refolded into monomeric soluble protein, indicating formation of stable independent domains. The homology model was used to predict putative substrate sequences. Homologs of mammalian CLCA genes were detected in the genomes of a vast array of multicellular animals: lower vertebrates, tunicates, insects, crustaceans, echinoderms, and flatworms. The hydrolase prediction is discussed in the context of published experimentally determined effects of CLCA proteins on chloride conductance. Altered proteolytic processing of full-length CLCA1 containing a mutation abolishing the predicted hydrolase activity is shown as initial experimental evidence for a role of the hydrolase domain in processing of mature full-length CLCA1. The hydrolase prediction together with the presented experimental data add to doubts about the function of CLCAs as chloride channels and strengthen the hypothesis of channel-activating and/or channel-accessory roles.

Amino Acid Sequence↗
PubMed1983

Isolation and characterization of monoclonal antibodies against rat liver epoxide hydrolase.

We have established hybridoma cell lines which secrete monoclonal antibody to rat liver microsomal epoxide hydrolase. All of the monoclonal antibodies formed against epoxide hydrolase are mouse immunoglobulin subclass IgG1. Utilizing double immunodiffusion analysis, we have found that the monoclonal antibodies bind to and precipitate epoxide hydrolase in solubilized rat liver microsomes. Despite the ability of the monoclonal antibodies to precipitate rat liver microsomal epoxide hydrolase, they do not inhibit the catalytic activity of the enzyme but rather stimulate it. The monoclonal antibodies do not precipitate epoxide hydrolase in microsomes isolated from hamster, rabbit, monkey, or human. When rabbit reticulocyte lysates are programmed with rat liver mRNA, the primary translation product of epoxide hydrolase can be immunoprecipitated from the translation system using the monoclonal antibodies. The ability of the antibodies to recognize in vitro synthesized epoxide hydrolase should make them amenable to identify recombinant bacterial clones expressing this protein. The monoclonality of these antibodies and their specificity should provide a useful way of identifying, purifying, and quantitating rat liver epoxide hydrolase as well as examine the expression of the gene(s) encoding epoxide hydrolase in normal rats and in rats exposed to carcinogenic xenobiotics.

Animals↗
PubMed1986

Distribution and inducibility of cytosolic epoxide hydrolase in male Sprague-Dawley rats.

Cytosolic epoxide hydrolase (cEH) activity has been determined in liver and various extrahepatic tissues of male Sprague-Dawley rats using trans-stilbene oxide (TSO) and trans-ethylstyrene oxide (TESO) as substrates. Large interindividual differences in the specific activity of cytosolic epoxide hydrolase in the liver from more than 80 individual rats were observed varying by a factor of 38. In a randomly selected group of five animals liver cEH varied by a factor of 3.9 and kidney cEH by a factor of 2.7, whereas liver microsomal epoxide hydrolase and lactate dehydrogenase showed only very low variations (1.4- and 1.1-fold, respectively). The individual relative activity of kidney cEH was related to that of the liver. Cytosolic epoxide hydrolase activity was present in all of six extrahepatic rat tissues investigated. Interestingly specific activities were very high in the heart and kidney (higher than in liver), followed by liver greater than brain greater than lung greater than testis greater than spleen. TSO and TESO hydrolases in subcellular fractions of rat liver were present at highest specific activities in the cytosolic and the heavy mitochondrial fraction. As indicated by the marker enzymes, catalase, urate oxidase and cytochrome oxidase, this organelle-bound epoxide hydrolase activity may be of peroxisomal and/or mitochondrial origin. In the microsomal fraction, TSO and TESO hydrolase activity is very low, whereas STO hydrolase activity is highest in this fraction and very low in cytosol. In kidney, subcellular distribution is similar to that observed in liver. None of the commonly used inducers of xenobiotic metabolizing enzymes caused significant changes in the specific activities of rat hepatic cEH (trans-stilbene oxide, alpha-pregnenolone carbonitrile, 3-methylcholanthrene, beta-naphthoflavone, isosafrole, butylated hydroxytoluene, 2,3,7,8-tetrachlorodibenzo-p-dioxin, dibenzo[a,h]anthracene, phenobarbitone). However, clofibrate, a hypolipidemic agent, very strongly induced rat liver cEH (about 5-fold), whereas microsomal epoxide hydrolase activity was not affected. Specific activity of kidney cEH was increased about 2-fold.

Animals↗
PubMed2006

Unique regulation of the active site of the serine esterase S-formylglutathione hydrolase.

S-Formylglutathione hydrolases (SFGHs) are highly conserved thioesterases present in prokaryotes and eukaryotes, and form part of the formaldehyde detoxification pathway, as well as functioning as xenobiotic-hydrolysing carboxyesterases. As defined by their sensitivity to covalent modification, SFGHs behave as cysteine hydrolases, being inactivated by thiol alkylating agents, while being insensitive to inhibition by organophosphates such as paraoxon. As such, the enzyme has been classified as an esterase D in animals, plants and microbes. While SFGHs do contain a conserved cysteine residue that has been implicated in catalysis, sequence analysis also reveals the classic catalytic triad of a serine hydrolase. Using a combination of selective protein modification and X-ray crystallography, AtSFGH from Arabidopsis thaliana has been shown to be a serine hydrolase rather than a cysteine hydrolase. Uniquely, the conserved reactive cysteine (Cys59) previously implicated in catalysis lies in close proximity to the serine hydrolase triad, serving a gate-keeping function in comprehensively regulating access to the active site. Thus, any covalent modification of Cys59 inhibited all hydrolase activities of the enzyme. When isolated from Escherichia coli, a major proportion of recombinant AtSFGH was recovered with the Cys59 forming a mixed disulfide with glutathione. Reversible disulfide formation with glutathione could be demonstrated to regulate hydrolase activity in vitro. The importance of Cys59 in regulating AtSFGH in planta was demonstrated in transient expression assays in Arabidopsis protoplasts. As determined by fluorescence microscopy, the Cys59Ser mutant enzyme was shown to rapidly hydrolyse 4-methylumbelliferyl acetate in paraoxon-treated cells, while the native enzyme was found to be inactive. Our results clarify the classification of AtSFGHs as hydrolases and suggest that the regulatory and conserved cysteine provides an unusual redox-sensitive regulation to an enzyme functioning in both primary and xenobiotic metabolism in prokaryotes and eukaryotes.

Amino Acid Sequence↗
PubMed2000

Epoxide hydrolases: biochemistry and molecular biology.

Epoxides are organic three-membered oxygen compounds that arise from oxidative metabolism of endogenous, as well as xenobiotic compounds via chemical and enzymatic oxidation processes, including the cytochrome P450 monooxygenase system. The resultant epoxides are typically unstable in aqueous environments and chemically reactive. In the case of xenobiotics and certain endogenous substances, epoxide intermediates have been implicated as ultimate mutagenic and carcinogenic initiators Adams et al. (Chem. Biol. Interact. 95 (1995) 57-77) Guengrich (Properties and Metabolic roles 4 (1982) 5-30) Sayer et al. (J. Biol. Chem. 260 (1985) 1630-1640). Therefore, it is of vital importance for the biological organism to regulate levels of these reactive species. The epoxide hydrolases (E.C. 3.3.2. 3) belong to a sub-category of a broad group of hydrolytic enzymes that include esterases, proteases, dehalogenases, and lipases Beetham et al. (DNA Cell Biol. 14 (1995) 61-71). In particular, the epoxide hydrolases are a class of proteins that catalyze the hydration of chemically reactive epoxides to their corresponding dihydrodiol products. Simple epoxides are hydrated to their corresponding vicinal dihydrodiols, and arene oxides to trans-dihydrodiols. In general, this hydration leads to more stable and less reactive intermediates, however exceptions do exist. In mammalian species, there are at least five epoxide hydrolase forms, microsomal cholesterol 5,6-oxide hydrolase, hepoxilin A(3) hydrolase, leukotriene A(4) hydrolase, soluble, and microsomal epoxide hydrolase. Each of these enzymes is distinct chemically and immunologically. Table 1 illustrates some general properties for each of these classes of hydrolases. Fig. 1 provides an overview of selected model substrates for each class of epoxide hydrolase.

Animals↗
PubMed1982

Purification of human liver cytosolic epoxide hydrolase and comparison to the microsomal enzyme.

Epoxide hydrolase (EC 3.3.2.3) was purified to electrophoretic homogeneity from human liver cytosol by using hydrolytic activity toward trans-8-ethylstyrene 7,8-oxide (TESO) as an assay. The overall purification was 400-fold. The purified enzyme has an apparent monomeric molecular weight of 58 000, significantly greater than the 50 000 found for human (or rat) liver microsomal epoxide hydrolase or for another TESO-hydrolyzing enzyme also isolated from human liver cytosol. Purified cytosolic TESO hydrolase catalyzes the hydrolysis of cis-8-ethylstyrene 7,8-oxide 10 times more rapidly than does the microsomal enzyme, catalyzes the hydrolysis of TESO and trans-stilbene oxide as rapidly as the microsomal enzyme, but catalyzes the hydrolysis of styrene 7,8-oxide, p-nitrostyrene 7,8-oxide, and naphthalene 1,2-oxide much less effectively than does the microsomal enzyme. Purified cytosolic TESO hydrolase does not hydrolyze benzo[a]pyrene 4,5-oxide, a substrate for the microsomal enzyme. The activities of the purified enzymes can explain the specific activities observed with subcellular fractions. Anti-human liver microsomal epoxide hydrolase did not recognize cytosolic TESO hydrolase in purified form or in cytosol, as judged by double-diffusion immunoprecipitin analysis, precipitation of enzymatic activity, and immunoelectrophoretic techniques. Cytosolic TESO hydrolase and microsomal epoxide hydrolase were also distinguished by peptide mapping. The results provide evidence that physically different forms of epoxide hydrolase exist in different subcellular fractions and can have markedly different substrate specificities.

Adolescent↗
PubMed1997

Isozyme-specific transition state inhibitors for the trypanosomal nucleoside hydrolases.

Protozoan parasites lack de novo purine biosynthesis and require purine salvage from the host. Nucleoside hydrolases are involved in nucleoside salvage and are not found in mammals, making them protozoan-specific targets for inhibitor design. Several protozoan nucleoside hydrolase isozymes with distinct substrate specificities have been characterized. Novel substituted iminoribitols have been synthesized to resemble the transition state structure of the nonspecific inosine-uridine nucleoside hydrolase from Crithidia fasciculata (IU-nucleoside hydrolase). These inhibitors have been characterized for this enzyme and for a purine-specific nucleoside hydrolase (IAG-nucleoside hydrolase) from Trypanosoma brucei brucei. Inhibitors which provide nanomolar inhibition constants for IU-nucleoside hydrolase exhibit micromolar inhibition constants for the IAG-enzyme. For example, p-bromophenyliminoribitol inhibits the IU- and IAG-enzymes with dissociation constants of 28 nM and 190 microM, respectively. Substrate specificity, the action of transition state inhibitors and the pH-dependence of the kinetic constants establish that the catalytic mechanisms and transition state structures are fundamentally different for the IU- and IAG-isozymes. The finding is remarkable since these isozymes share significant homology at the catalytic sites and both use inosine as a preferred substrate. The specificity of the transition state analogues indicates that logically-designed transition state inhibitors are isozyme-specific, with (Km/Ki IU-nucleoside hydrolase)/(Km/Ki IAG-nucleoside hydrolase) values up to 39,000. The mechanism of the differential inhibition is based on the relative leaving group activation and ribosyl-oxocarbenium-forming abilities of these enzymes. In addition to providing isozyme-specific inhibitors, the novel molecules described here have diagnostic value for the nature of the transition states for N-ribohydrolase enzymes.

Animals↗
PubMed1992

Leukotriene A4 hydrolase: abrogation of the peptidase activity by mutation of glutamic acid-296.

The metal-binding motif in the sequence of leukotriene A4 (LTA4) (EC 3.3.2.6), a bifunctional zinc metalloenzyme, contains a glutamic acid that is conserved in several zinc hydrolases. To study its role for the two catalytic activities, Glu-296 in mouse leukotriene A4 hydrolase was replaced by a glutamine or alanine residue by site-directed mutagenesis. Wild-type and mutated cDNAs were expressed four or five times in Escherichia coli, and the resulting proteins were purified to apparent homogeneity. With respect to their epoxide hydrolase activities--i.e., the conversion of LTA4 into leukotriene B4--the mutated enzymes [Gln296]LTA4 hydrolase and [Ala296]LTA4 hydrolase exhibited specific activities of 1070 +/- 160 and 90 +/- 30 nmol of LTB4 per mg of protein per min (mean +/- SD; n = 4 or 5), respectively, corresponding to 150% and 15% of unmutated enzyme. In contrast, when the mutated proteins were assayed for peptidase activity toward alanine-4-nitroanilide, they were found to be virtually inactive (less than or equal to 0.2% of unmutated enzyme). To serve as a positive control, we also replaced Ser-298 with an alanine residue, which resulted in a protein ([Ala298]LTA4 hydrolase) with catalytic properties almost indistinguishable from the wild-type enzyme. Substitution of Glu-296 by glutamine or alanine was also carried out with human LTA4 hydrolase, and the mutated human enzymes displayed specific activities similar to the corresponding mouse proteins. Zinc analyses of the purified mouse and human proteins confirmed that the mutations did not significantly influence their zinc content. In conclusion, the results of the present study indicate a direct catalytic role for Glu-296 in the peptidase reaction of LTA4 hydrolase, where it presumably acts as a base to polarize water, whereas its function, if any, is apparently not essential in the epoxide hydrolase reaction.

Amino Acid Sequence↗
PubMed1997

Primary structure and catalytic mechanism of the epoxide hydrolase from Agrobacterium radiobacter AD1.

The epoxide hydrolase gene from Agrobacterium radiobacter AD1, a bacterium that is able to grow on epichlorohydrin as the sole carbon source, was cloned by means of the polymerase chain reaction with two degenerate primers based on the N-terminal and C-terminal sequences of the enzyme. The epoxide hydrolase gene coded for a protein of 294 amino acids with a molecular mass of 34 kDa. An identical epoxide hydrolase gene was cloned from chromosomal DNA of the closely related strain A. radiobacter CFZ11. The recombinant epoxide hydrolase was expressed up to 40% of the total cellular protein content in Escherichia coli BL21(DE3) and the purified enzyme had a kcat of 21 s-1 with epichlorohydrin. Amino acid sequence similarity of the epoxide hydrolase with eukaryotic epoxide hydrolases, haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, and bromoperoxidase A2 from Streptomyces aureofaciens indicated that it belonged to the alpha/beta-hydrolase fold family. This conclusion was supported by secondary structure predictions and analysis of the secondary structure with circular dichroism spectroscopy. The catalytic triad residues of epoxide hydrolase are proposed to be Asp107, His275, and Asp246. Replacement of these residues to Ala/Glu, Arg/Gln, and Ala, respectively, resulted in a dramatic loss of activity for epichlorohydrin. The reaction mechanism of epoxide hydrolase proceeds via a covalently bound ester intermediate, as was shown by single turnover experiments with the His275 --> Arg mutant of epoxide hydrolase in which the ester intermediate could be trapped.

Amino Acid Sequence↗
PubMed2001

On the role of conserved histidine 106 in 10-formyltetrahydrofolate dehydrogenase catalysis: connection between hydrolase and dehydrogenase mechanisms.

The enzyme, 10-formyltetrahydrofolate dehydrogenase (FDH), converts 10-formyltetrahydrofolate (10-formyl-THF) to tetrahydrofolate in an NADP(+)-dependent dehydrogenase reaction or an NADP(+)-independent hydrolase reaction. The hydrolase reaction occurs in a 310-amino acid long amino-terminal domain of FDH (N(t)-FDH), whereas the dehydrogenase reaction requires the full-length enzyme. The amino-terminal domain of FDH shares some sequence identity with several other enzymes utilizing 10-formyl-THF as a substrate. These enzymes have two strictly conserved residues, aspartate and histidine, in the putative catalytic center. We have shown recently that the conserved aspartate is involved in FDH catalysis. In the present work we studied the role of the conserved histidine, His(106), in FDH function. Site-directed mutagenesis experiments showed that replacement of the histidine with alanine, asparagine, aspartate, glutamate, glutamine, or arginine in N(t)-FDH resulted in expression of insoluble proteins. Replacement of the histidine with another positively charged residue, lysine, produced a soluble mutant with no hydrolase activity. The insoluble mutants refolded from inclusion bodies adopted a conformation inherent to the wild-type N(t)-FDH, but they did not exhibit any hydrolase activity. Substitution of alanine for three non-conserved histidines located close to the conserved one did not reveal any significant changes in the hydrolase activity of N(t)-FDH. Expressed full-length FDH with the substitution of lysine for the His(106) completely lost both the hydrolase and dehydrogenase activities. Thus, our study showed that His(106), besides being an important structural residue, is also directly involved in both the hydrolase and dehydrogenase mechanisms of FDH. Modeling of the putative hydrolase catalytic center/folate-binding site suggested that the catalytic residues, aspartate and histidine, are unlikely to be adjacent to the catalytic cysteine in the aldehyde dehydrogenase catalytic center. We hypothesize that 10-formyl-THF dehydrogenase reaction is not an independent reaction but is a combination of hydrolase and aldehyde dehydrogenase reactions.

Amino Acid Sequence↗
PubMed2005

Leukotriene A4 hydrolase, insights into the molecular evolution by homology modeling and mutational analysis of enzyme from Saccharomyces cerevisiae.

Mammalian leukotriene A4 (LTA4) hydrolase is a bifunctional zinc metalloenzyme possessing an Arg/Ala aminopeptidase and an epoxide hydrolase activity, which converts LTA4 into the chemoattractant LTB4. We have previously cloned an LTA4 hydrolase from Saccharomyces cerevisiae with a primitive epoxide hydrolase activity and a Leu aminopeptidase activity, which is stimulated by LTA4. Here we used a modeled structure of S. cerevisiae LTA4 hydrolase, mutational analysis, and binding studies to show that Glu-316 and Arg-627 are critical for catalysis, allowing us to a propose a mechanism for the epoxide hydrolase activity. Guided by the structure, we engineered S. cerevisiae LTA4 hydrolase to attain catalytic properties resembling those of human LTA4 hydrolase. Thus, six consecutive point mutations gradually introduced a novel Arg aminopeptidase activity and caused the specific Ala and Pro aminopeptidase activities to increase 24 and 63 times, respectively. In contrast to the wild type enzyme, the hexuple mutant was inhibited by LTA4 for all tested substrates and to the same extent as for the human enzyme. In addition, these mutations improved binding of LTA4 and increased the relative formation of LTB4, whereas the turnover of this substrate was only weakly affected. Our results suggest that during evolution, the active site of an ancestral eukaryotic zinc aminopeptidase has been reshaped to accommodate lipid substrates while using already existing catalytic residues for a novel, gradually evolving, epoxide hydrolase activity. Moreover, the unique ability to catalyze LTB4 synthesis appears to be the result of multiple and subtle structural rearrangements at the catalytic center rather than a limited set of specific amino acid substitutions.

Amino Acid Substitution↗
PubMed1984

Hydrolase secretion is a consequence of membrane recycling.

Acanthamoeba releases lysosomal hydrolases continuously into the culture medium. This release is specific for lysosomal hydrolases, but not other cellular proteins, and is energy dependent. The secreted hydrolases can be separated into two groups on the basis of their secretion kinetics: one is secreted at approximately 15% of the cellular activity per hour and the other at approximately 5%. Intracellularly the lysosomal hydrolases are restricted almost exclusively to secondary lysosomes where the hydrolases demonstrate a differential pH-dependent binding to membrane. Hydrolase secretion is not the result of secondary lysosomes' fusing with the plasma membrane since soluble and particulate lysosomal contents are not released at the same rate. Together the data suggest that the secreted hydrolases are trapped in shuttle vesicles that cycle membrane from secondary lysosomes to the cell surface. The inner membrane and content of these vesicles undergo a marked pH shift when, following fragmentation from lysosomes, these vesicles fuse with plasma membrane. This rapid pH shift and the differential pH-dependent membrane binding of hydrolases appear to account for the heterogeneous hydrolase secretion kinetics.

Amoeba↗
PubMed1998

2,3-epoxy-4-hydroxynonanal, a potential lipid peroxidation product for etheno adduct formation, is not a substrate of human epoxide hydrolase.

Our previous studies have shown that 2,3-epoxy-4-hydroxynonanal, a reactive epoxy aldehyde capable of forming etheno adducts with DNA bases, is mutagenic and tumorigenic (Carcinogenesis, 14, 2073). The epoxy aldehyde can be generated from trans-4-hydroxy-2-nonenal, a lipid peroxidation product of omega-6 polyunsaturated fatty acids, by autoxidation or by incubation with fatty acid hydroperoxides or hydrogen peroxides (Chem. Res. Toxicol., 9, 306). These are plausible in vivo pathways for the formation of 2,3-epoxy-4-hydroxynonanal. The possibility that 2,3-epoxy-4-hydroxynonanal is a tumorigen of endogenous origin is suggested by recent observations that etheno bases are detected as background DNA lesions in untreated rodents and humans. A metabolic pathway critical for detoxification of 2,3-epoxy-4-hydroxynonanal involves the ring-opening by epoxide hydrolase, which abolishes its ability to form cyclic etheno DNA adducts. In this study, we examined whether 2,3-epoxy-4-hydroxynonanal is a substrate of cDNA expressed human epoxide hydrolase. Human epoxide hydrolase was expressed in TK- 143 cells (thymidine kinase-deficient human embryoblast) infected with recombinant vaccinia virus encoding human epoxide hydrolase cDNA. Controls consisted of the cells infected with vaccinia virus in the absence of human epoxide hydrolase cDNA. No hydrolysis occurred when [2,3-(3)H]2,3-epoxy-4-hydroxynonanal was incubated at 37 degrees C for 30 min at pH 7.4 with cells expressing human epoxide hydrolase, as indicated by the presence of a pair of radioactive peaks in reversed-phase HPLC chromatography, which comigrated with the UV standards of the two diastereomers of the epoxy aldehyde. The identity of these compounds as the intact epoxy aldehyde was further supported by derivatization to the 2,4-dinitrophenylhydrazones followed by reversed phase HPLC analysis. Similar results were observed with the control cells or with the heat deactivated human epoxide hydrolase. The epoxide hydrolase activity in the expressed cells was demonstrated by their ability to convert benzo[a]pyrene-4,5-dihydroepoxide to benzo[a]pyrene-trans-4,5-dihydrodiol under the same conditions. These results clearly indicate that 2,3-epoxy-4-hydroxynonanal is not a substrate of human epoxide hydrolase, and, thus strengthen its possible endogenous role in the formation of promutagenic exocyclic etheno adducts in vivo.

Aldehydes↗
PubMed1982

Effect of hepatocarcinogens on epoxide hydrolase and other xenobiotic metabolizing enzymes.

Hepatocarcinogens cause marked biochemical changes in the liver at short intervals after administration. The studies described were designed to investigate the effects of hepatocarcinogens and hepatotoxicants on the microsomal mixed function oxidase system. DT-diaphorase and epoxide hydrolase. Following 5 day p.o. treatment of male F-344 rats with aflatoxin B1 (AFB), 2-acetylaminofluorene (AAF), technical grade dinitrotoluene (DNT), or 2,4-diaminotoluene, microsomal cytochrome P450 dependent enzyme activities were depressed while epoxide hydrolase activity was markedly elevated (3-8 times control). Diethylnitrosamine (DEN) given at 5 mg/kg/day and DL-ethionine at 1000 mg/kg/day failed to increase epoxide hydrolase. 3-Methylcholanthrene, methylnitrosourea, carbon tetrachloride, bromobenzene and vinyl chloride all failed to increase epoxide hydrolase activity. Using 3 daily i.p. injections, dose-response relationships for increases in epoxide hydrolase were generated for the hepatocarcinogens. With the exception of p-dimethylaminoazobenzene (DAB) and DEN, the carcinogens studied produced log-linear dose response curves for increase in epoxide hydrolase. Both DEN and DAB caused increases in epoxide hydrolase but classical sigmoidal dose-response curves were not obtained. The order of potency for increasing epoxide hydrolase was AFB greater than AAF greater than 2,6-dinitrotoluene greater than 3'-methyl-N,N-dimethyl-4-aminoazobenzene greater than DNT greater than 2, 4-dinitrotoluene. The slopes of the linear portions of the log dose-response curves were not statistically different from the slope of the dose-response curve obtained with AAF suggesting that structurally diverse carcinogens elicit increases in epoxide hydrolase by a common mechanism.

Animals↗