Term
| What is the main function of epoxide hydrolase? |
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Definition
| To add water to an oxirane ring to form a dihydrodiol |
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Term
| What is the difference between ester hydrolase and epoxide hydrolase? |
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Definition
Ester hydrolase splits the molecule apart.
Eposide hydrolase just adds water to the oxirane ring. |
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Term
| Epoxides are key intermediates formed during cytochorome-P450 mediated oxygenation of what? |
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Definition
| Double bonds and aromatic rings |
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Term
| What are the two forms of EH? |
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Definition
mEH - microsomal form shown to convert potentially toxic epoxides to dihydrodiols (i.e. activation of benzo(a)pyrene to reactive diol-epoxide.
sEH - soluble form |
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Term
Fill in the blanks:
Although epoxides, as strained 3-membered rings, are chemically reactive ________ and readily attack ________ such as water, the rate of hydrolysis is increased by ____________. |
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Definition
Electrophiles
Nucleophiles
Epoxide Hydrolase Enzyme |
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Term
| Do we see non-enzymatic hydrolysis of epoxides? |
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Definition
| Yes, because the epoxide ring is strained.. however EH increases this rate. |
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Term
| What type of oxirane ring is formed when EH attacks a d.b.? |
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Definition
Alkene Oxide
examples styrene oxide and octene oxide |
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Term
| Do EH's usually lead to toxic compounds? |
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Definition
| No, it is generally a detox rxn because it takes away the electrophile.. however. Sometimes, it can increase toxicity (In BaP) |
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Term
| Describe the role of mEH in the toxicity of benzo(a)pyrene. |
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Definition
| First, CYPIA forms an epoxide at the 7,8 bond of the rignt o form Benzo(a)pyrene-7,8-oxide. mEH attacks the oxide ring to give a 7,8-dihydrodiol. This can undergo another round of oxidation by CYP1A to vite the anti-diol-epoxide (a hightly toxic metabolite. This second epoxide is not a good substrate for EH and will not be hydrolized to non-toxic tetrol. It will instead bind to cellular nucleophiles including DNA (carcinogenicity) and protein and RNA. |
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Term
| How many forms are there of EH in most animals? |
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Definition
| In most animals, there's only one form of each, mEH and sEH. |
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Term
| Describe substrates for mEH? Give an example. |
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Definition
mEH uses xenobiotic cis-eposides as substrates
e.g. cis-stilbene oxide |
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Term
| Describe substrates for sEH. Give an example. |
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Definition
Cytosolic epooside hydrolase mEH sues xenobiotic trans-eposides as stubstrates.
e.g. trans-stilbene oxide |
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Term
| Describe the polypeptide chain of mEH? |
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Definition
| The single microsomal form has a MW of ~51,000 - close to P450 |
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Term
| Where is the N-terminus of mEH anchored? |
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Definition
| N-terminal portion anchors mEH oprotein to the endoplasmic reticulum membrane (towards cytsol in liver, lung, intestine cells, etc.) |
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Term
| In what tissues do we find mEH proteins? |
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Definition
| Liver, lung and other tissues (intestine) |
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Term
| Which EH (mEH or sEH) is oriented towares the cell cytosol, and not the lumen or the ER? |
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Definition
| mEH - N-term is anchored to ER |
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Term
| Describe the polypeptide chain of sEH. |
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Definition
| Trans-epoxide metabolizing sEH exists in solution as a homodimer with a momomeric MW of 62,000. |
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Term
| Describe the hydrolysis of eposyeicosatrienoic acids (EETs) to dihydrodiols. |
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Definition
| sEH can metabolise important trans epoxides such as EETs into dihydrodiols (DHETs), which regulate blood pressure. It is thought that an inhibition of this enzyme could be a useful drug for keeping BP low. |
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Term
| Do mEH and sEH have the same aa sequence? |
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Definition
No, in fact, comparison of sEH and mEH from a single species shows no obvious similarity.
Active site residues important in catalysis are fairly similar, but the N-term of mEH has a hydrophobic part anchored to the ER membrane (which is not found in the solubulized form. |
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Term
| What is the bacterial haloalkane dehalogenase? |
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Definition
An enzyme that showed similarity to mEH and sEH... these three make up the alpha-beta hydrolase fold enzyme family.
Both soluble and microsomal forms of EH are part of the larger haloalkane dehalogenase related alpha-beta hydrolase family. |
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Term
| Why are sEH, mEH, and bacterial haloalkane dehalogenase all apart of the same family? |
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Definition
| They all have a central beta-sheet flanked by alpha helices. (alpha-beta hydrolase fold enzyme family) |
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Term
| Describe how they were able to determine the mechanism of hydrolysis in EH? |
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Definition
It was shown, in the presence of 18O labeled H2O, that a single rate of substrate of substrate turnover lead to the 18O being incorporated into the enzyme (rather than the substrate), which suggested the formation of an enzyme-substrate ester formation.
Because of the active-site sequence similarities between sEH and mEH, they were able to conclude that they both were forming an enzyme-substrate ester intermediate. |
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Term
| Describe the mechanism of hydrolysis for the alpha-beta hydrolase enzyme Superfamily. |
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Definition
| An acidic group in the enzyme active site attacks the oxirane ring to form an ester-type interemediate in which the substrate is bound to the enzyme. Then a base at another part of the enzyme deprotonates water, and the hydroxy group attacks the oxirane ring. (works as a catalytic triad) |
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Term
| Describe the role of the two tyrosine residues in mEH and sEH. |
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Definition
| The OH groups of the 2 tyrosines located 'across' from the active site each form a hydrogen bond with the oxygen of the oxirane ring. This keeps it close to the active Aspartate (Asp 333) and therefore facilitates the formation of the ester-intermediate |
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Term
| After the formation of the enzyme-substrate ester intermediate (once the aspartic acid Asp333 has attacked the oxirane ring), describe the functions of the Asp495 and His523, which make up the rest of this catalytic triad? |
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Definition
The aspartic acid deprotonates the histidine, which in turn deprotonates water, which makes the hydroxyl active enough to hydrolyze to form the dihydrodiol.
(mechanism for mEH and sEH) |
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Term
| Describe how trans-stilbene oxide interacts with epoxide hydrolase enzymes. |
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Definition
Trans-stilbene oxide is a good inducer of mEH (up to a 7-fold increase of expression).
For sEH - trans-stilbene epoxides are susbrates and can react to form the dihydrodiol. |
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Term
| How does phenobarbital effect EH's? |
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Definition
| It induces mEH 2-3 fold (along with imidazole derivatives and peroxisome proliferators) |
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Term
| How do imidazole derivites effect EH's? |
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Definition
| Imidazole derivatives induce mEH 2-3 fold. |
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Term
| How do peroxisome proliferators effect EH's? |
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Definition
| Peroxisome proliferators induce mEH 2-3 fold. |
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Term
| What is acetylaminofluorene and how does it effect EH activity? |
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Definition
Acetylaminofluorine is a carcinogen which upregulates mEH greatly (7-fold).
There is some evidence that other carcinogens can upregulate mEH as well. |
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Term
| What are the two major inducers of mEH? What is the only inducer of sEH? |
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Definition
trans-stilbene oxide and acetylaminofluorine cause 7-fold induction for mEH
sEH is only inucible by perosisome proliferating agents |
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Term
| What is TCPO (1,1,1-trichloropropene oxide)? |
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Definition
| A competitive inhibitor of mEH. It is a poor mEH substrate and will occupy the active site and prevent other epoxides from binding. |
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Term
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Definition
Valpromide inhibits mEH...
It contains an amine group
Carbonyl forms H bonds with tyrosine
Amino group form H bonds with aspartate
(sits in active site and prents hydrolysis of subsequent substrates) |
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Term
| How do 1-benzylimidazole and clotrimazole effect enzymatic activity in the cell? |
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Definition
| Both contain imidizole groups which inhibit P450, yet activate mEH. |
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Term
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Definition
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Term
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Definition
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Term
| How to chalcone oxides and their derivatives effect EH activity? |
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Definition
| chalcone oxides enhance activity of mEH, but their derivatives (such as 4-fluoro-chalcone oxide) can be competitive inhibitors of sEH (potential BP regulator) |
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Term
| What is the function of urea derivative (N-cyclohexyl-N'(3-phenylpropyl)urea on EH activity? |
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Definition
| This urea derivative inhibits sEH. (possible BP regulator) |
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Term
| How does treatment of 3MC affect napthalene and anthracene metabolism? |
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Definition
| Napthalene and anthracene are polycyclic aromatic hydrocarbons (potential carcinogenic substances. If rat microsomes were pretreated with 3MC (which induces CYP1A), a very highly steroselective form of the dihydrodiol was seen. |
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Term
| How does treatement of phenobarbital affect the metabolism of napthalene and anthracene? |
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Definition
| Napthalene and anthracene are polycyclic aromatic hydrocarbons (potential carcinogenic substances. If rat microsomes were pretreated with phenobarbital (which induces CYP2B), there would be a nubmer opf stereoisomers formed (not stereoselective). |
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Term
| When it was seen that 3MC treated microsomes had a stereoselective formation of the dihydrodiol, what did it suggest? |
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Definition
It suggested a coupling between CYP1A and microsomal epoxide hydrolase (mEH).
This could be advantagous if we have a carcinogenic compound that would go to the less toxic dihydrodiol... |
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Term
| How can you explain the strong stereoselectivity of dihydrodiol formation in 3MC treated microsomes? |
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Definition
3MC induces CYP1A, and leads to a lower Km - a faster hydrolysis. CYP1A leads to a better substrate for epoxide hydrolases, which means it will be hydrolysed right away and will not likely undergo any non-enzymatic or alternative enzymatic hydrolysis.
Alternatively, PB treated microsomes induce CYP2B. Their epoxide products have a higher Km and are much less efficent substrates. These epoxides have the chance to leave the cell and be metabolized by different enzymes (or even non-enzymatic hydrolysis).. this leads to many forms of the dihydrodiol. |
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