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Catalysis of potato epoxide hydrolase, StEH1.

ABSTRACT: The kinetic mechanism of epoxide hydrolase (EC 3.3.2.3) from potato, StEH1 (Solanum tuberosum epoxide hydrolase 1), was studied by presteady-state and steady-state kinetics as well as by pH dependence of activity. The specific activities towards the different enantiomers of TSO (trans-stilbene oxide) as substrate were 43 and 3 micromol x min(-1) x mg(-1) with the R,R- or S,S-isomers respectively. The enzyme was, however, enantioselective in favour of the S,S enantiomer due to a lower K(m) value. The pH dependences of kcat with R,R or S,S-TSO were also distinct and supposedly reflecting the pH dependences of the individual kinetic rates during substrate conversion. The rate-limiting step for TSO and cis- and trans-epoxystearate was shown by rapid kinetic measurements to be the hydrolysis of the alkylenzyme intermediate. Functional characterization of point mutants verified residues Asp105, Tyr154, Tyr235 and His300 as crucial for catalytic activity. All mutants displayed drastically decreased enzymatic activities during steady state. Presteady-state measurements revealed the base-deficient H300N (His300-->Asn) mutant to possess greatly reduced efficiencies in catalysis of both chemical steps (alkylation and hydrolysis).

SUBMITTER: Elfstrom LT

PROVIDER: S-EPMC1198943 | biostudies-other | 2005 Sep

REPOSITORIES: biostudies-other

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Conformational diversity and enantioconvergence in potato epoxide hydrolase 1.

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Project description:A series of N,N'-disubstituted ureas having a conformationally restricted cis- or trans-1,4-cyclohexane alpha to the urea were prepared and tested as soluble epoxide hydrolase (sEH) inhibitors. This series of compounds showed low nanomolar to picomolar activities against recombinant human sEH. Both isomers showed similar potencies, but the trans isomers were more metabolically stable in human hepatic microsomes. Furthermore, these new potent inhibitors show a greater metabolic stability in vivo than previously described sEH inhibitors. We demonstrated that trans-4-[4-(3-adamantan-1-ylureido)cyclohexyloxy]benzoic acid 13g (t-AUCB, IC50 = 1.3 +/- 0.05 nM) had excellent oral bioavailability (98%, n = 2) and blood area under the curve in dogs and was effective in vivo to treat hypotension in lipopolysaccharide challenged murine models.

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Project description:A series of inhibitors of the soluble epoxide hydrolase (sEH) containing one or two thiourea groups has been developed. Inhibition potency of the described compounds ranges from 50 μM to 7.2 nM. 1,7-(Heptamethylene)bis[(adamant-1-yl)thiourea] (6f) was found to be the most potent sEH inhibitor, among the thioureas tested. The inhibitory activity of the thioureas against the human sEH is closer to the value of activity against rat sEH rather than murine sEH. While being less active, thioureas are up to 7-fold more soluble than ureas, which makes them more bioavailable and thus promising as sEH inhibitors.

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Project description:Neuroinflammation has been increasingly recognized to play a critical role in Alzheimer’s disease (AD). The epoxy fatty acids (EpFAs) are derivatives of the arachidonic acid metabolism pathway and have anti-inflammatory activities. However, their efficacy is limited due to their rapid hydrolysis by the soluble epoxide hydrolase (sEH). We report that sEH is predominantly expressed in astrocytes and its concentrations are elevated in postmortem brain tissue from AD patients and in the 5xFAD -amyloid mouse model of AD. The amount of sEH expressed in AD mouse brains correlated with a reduction in brain EpFA concentrations. Using a specific small molecule sEH inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), we report that TPPU treatment protected AD mice against LPS-induced inflammation in vivo. Long-term administration of TPPU to the 5xFAD mouse model via drinking water reversed microglia and astrocyte reactivity and immune pathway dysregulation. This was associated with reduced β-amyloid pathology and improved synaptic integrity and cognitive function on two behavioral tests. Importantly, TPPU treatment correlated with an increase in EpFA concentrations in the brains of 5xFAD mice, demonstrating brain penetration and target engagement. These findings support further investigation of TPPU as a potential therapeutic agent for the treatment of AD.

2020-11-20 | GSE161848 | GEO

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