Leukotriene A4 hydrolase: determination of the three zinc-binding ligands by site-directed mutagenesis and zinc analysis.

Medina, Juan F.; Wetterholm, Anders; Rådmark, Olof; Shapiro, Robert; Haeggström, Jesper Z.; Vallée, Bert L.; Samuelsson, Bengt

doi:10.1073/pnas.88.17.7620

Cited by 111 publications

(76 citation statements)

References 28 publications

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“…4B). Although aspartate can be a zinc ligand (Le Moual et al 1991,Medina et al 1991,Vallee & Auld 1990b,Vallee & Auld 1990a), it does not appear to function this way in ASPA. The E24D mutation in ASPA showed no detectable activity from either crude COS-7 cell extract or when partially purified using nickel-affinity chromatography of a C-terminal V5-His tag (data not shown).…”

Section: Characterization Of Zinc-coordination and Aspa Catalysismentioning

confidence: 99%

Mutational analysis of aspartoacylase: Implications for Canavan Disease

et al. 2007

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Mutations that result in undetectable activity of aspartoacylase, which catalyzes the deacetylation of N-acetyl-L-aspartate, correlate with Canavan Disease, a neurodegenerative disorder usually fatal during childhood. The underlying biochemical mechanisms of how these mutations ablate activity are poorly understood. Therefore, we developed and tested a three-dimensional homology model of aspartoacylase based on zinc dependent carboxypeptidase A. Mutations of the putative zinc-binding residues (H21G, E24D/G, and H116G), the general proton donor (E178A), and mutants designed to switch the order of the zinc-binding residues (H21E/E24H and E24H/H116E) yielded wild-type aspartoacylase protein levels and undetectable ASPA activity. Mutations that affect substrate carboxyl binding (R71N) and transition state stabilization (R63N) also yielded wild-type aspartoacylase protein levels and undetectable aspartoacylase activity. Alanine substitutions of Cys124 and Cys152, residues indicated by homology modeling to be in close proximity and in the proper orientation for disulfide bonding, yielded reduced ASPA protein and activity levels. Finally, expression of several previously tested (E24G, D68A, C152W, E214X, D249V, E285A, and A305E) and untested (H21P, A57T, I143T, P183H, M195R, K213E/G274R, G274R, and F295S) Canavan Disease mutations resulted in undetectable enzyme activity, and only E285A and P183H showed wild-type aspartoacylase protein levels. These results show that aspartoacylase is a member of the caboxypeptidase A family and offer novel explanations for most loss-of-function aspartoacylase mutations associated with Canavan Disease.

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Section: Characterization Of Zinc-coordination and Aspa Catalysismentioning

confidence: 99%

Mutational analysis of aspartoacylase: Implications for Canavan Disease

et al. 2007

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“…We therefore conclude that cholesterol EH is probably mechanistically and thus structurally unrelated to microsomal EH and soluble EH and belongs to a separate class of hydrolytic enzymes. Further insight into its structure and/or mechanism has to be obtained to elucidate its possible relationship to other enzymes, such as the leukotriene-A4 hydrolase (Medina et al, 1991). …”

Section: Discussionmentioning

confidence: 99%

Visualization of a Covalent Intermediate between Microsomal Epoxide Hydrolase, but not Cholesterol Epoxide Hydrolase, and their Substrates

Müller

Arand

Frank

et al. 1997

European Journal of Biochemistry

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Mammalian soluble and microsomal epoxide hydrolases have been proposed to belong to the family of n/~-hydrolase-fold enzymes. These enzymes hydrolyse their substrates by a catalytic triad, with the first step of the enzymatic reaction being the formation of a covalent enzyme-substrate ester. In the present paper, we describe the direct visualization of the ester formation between rat microsomal epoxide hydrolase and its substrate. Microsomal epoxide hydrolase was precipitated with acetone after brief incubation with 11 -"C]epoxystearic acid. After denaturing SDS gel electrophoresis the protein-bound radioactivity was detected by fluorography. Pure epoxide hydrolase and crude microsomes showed a single radioactive signal of the expected molecular mass that could be suppressed by inclusion of the competitive inhibitor 1 ,I ,I -trichloropropene oxide in the incubation mixture. In a similar manner, 4-fluorochalconeoxide-senaitive binding of epoxystearic acid to rat soluble epoxide hydrolase could be demonstrated in rat liver cytosol. Under similar conditions, no covalent binding of [26-'JC]cholesterol-5a,6n-epoxide to microsomal proteins or solubilized fractions tenfold enriched in cholesterol epoxide hydrolase activity could be observed. Our data provide definitive proof for the formation of an enzyme-substrate-ester intermediate formed in the course of epoxide hydrolysis by microsomal epoxide hydrolase, show no formation of a covalent intermediate between cholesterol epoxide hydrolase and its substrate under the same conditions as those under which an intermediate was shown for both microsomal and soluble epoxide hydrolases and therefore indicate that the cholesterol epoxide hydrolase apparently does not act by a similar mechanism and is probably not structurally related to microsomal and soluble epoxide hydrolases.Keywords ; epoxide hydrolase ; mechanism ; a/b hydrolase fold ; cholesterol ; fatty acid metabolism.Epoxide hydrolases (EH) represent a group of ubiquitous enzymes with important functions in the detoxification of reactive intermediates, namely epoxides, that arise from a large variety of compounds during their metabolism. The two mammalian enzymes implicated in the metabolism of foreign compounds are microsomal EH (Oesch, 1973) and soluble EH (Ota and Hammock, 1980). Both enzymes have been cloned from a variety of species (Beetham et al., 1993;Grant et al., 1993;Jackson et al., 1987;Knehr et al., 1993;Porter et al., 1986;Wojtasek and Prestwich, 1996). A third enzyme, cholesterol EH, which is membrane bound similarly to inicrosomal EH but otherwise distinct from the latter (Oesch et al., 1984), is less well investigated. Its physiological function appears to be the conversion of 5n,6n-epoxycholestane-3P-o1 and SP,6D-epoxycholestane-3/!-ol, the two epoxides arising from cholesterol during, e.g. lipid peroxidation, to give the single product cholestane-3~,Scr,b/j-triol (Watabe et al., 1981).For a long time it was believed that EH convert their substrates by direct hydrolysis. The pioneering work of Hanzli...

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“…Primers used for the construction of [E296Q] and [S298A]LTA % hydrolase were as described in [11]. Mutated proteins were expressed in E. coli (JM 101) transformed with the corresponding mutated plasmid, as described [15]. Sequence analysis of the entire cDNA inserts confirmed that no alterations of the protein primary structure, other than the desired mutation, had occurred.…”

Section: Site-directed Mutagenesis Of Lta 4 Hydrolase Cdna and Expresmentioning

confidence: 99%

“…Primers A and B were JF21 and JF27 [15]. Primer C was the mutagenetic primer in each case (site mutation underlined) : JF37 (5h-GACATAT-CTCATAGCTGGACAGG-3h) for Asp-296 ; JF38 (5h-AACAT-ATCTCATAGCTGGACAGG-3h) for Asn-296, both phosphorylated at the 5h end.…”

Section: Site-directed Mutagenesis Of Lta 4 Hydrolase Cdna and Expresmentioning

confidence: 99%

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Leukotriene A4 hydrolase: a critical role of glutamic acid-296 for the binding of bestatin

Andberg

Wetterholm

Medina³

et al. 2000

Biochemical Journal

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Leukotriene A4 hydrolase is a bifunctional Zn2+-containing enzyme catalysing the formation of the potent chemotaxin leukotriene B4. From an analysis of three mutants of Glu-296 we have found that this catalytic residue is critical for the binding of bestatin, a classical aminopeptidase inhibitor. For bestatin, but not for three other tight-binding inhibitors, the IC50 values for inhibition of the epoxide hydrolase activity decreased in the mutants to 0.7-0.003% of the control. Hence Glu-296 is an important structural determinant for binding of bestatin to leukotriene A4 hydrolase; this conclusion might also apply to other members of the M1 family of metallopeptidases.

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Leukotriene A4 hydrolase: determination of the three zinc-binding ligands by site-directed mutagenesis and zinc analysis.

Cited by 111 publications

References 28 publications

Mutational analysis of aspartoacylase: Implications for Canavan Disease

Mutational analysis of aspartoacylase: Implications for Canavan Disease

Visualization of a Covalent Intermediate between Microsomal Epoxide Hydrolase, but not Cholesterol Epoxide Hydrolase, and their Substrates

Leukotriene A4 hydrolase: a critical role of glutamic acid-296 for the binding of bestatin

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