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155 Cards in this Set
- Front
- Back
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What form of the P450 complex gives the characteristic 450 nm absorbance? |
"Fe(2+) –CO |
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diagram the difference between the distal and proximal sides of the heme |
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Draw The Oxidized, intermediate, and Reduced forms of flavins
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draw the p450 cycle |
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FMO5 has little/no activity towards cannonical FMO substrates, what does it do?
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It has been reported to be a bayer villagerase(it inserts oxygen adjacent to a carbonyl in a ring expansion reaction) |
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How do you selectively inhibit p450's? |
P450 reductase antibodyMechanism based inhibitor (1-aminobenzotriazole "ABT")
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How is uncoupling measured
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by measuring the stoicheometries of the product
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Kinetic Isotope Effect
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A ratio of 2 rate constants for the identical reaction of 2 compounds that only differ in isotopic substitution
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Match the isoform with it's preferred substrate type:2E1, 2C9, 1A2, 2B6, 2D6, 3A4, 2A6Big, Small, Basic, Acidic, Flat, Bent, Medium Planarity
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2E1 - Small3A4 - Big2C9 Acidic2D6 - Basic2B6 - Bent1A2 - Flat2A6 - Medium Planarity |
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Regioselectivity
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relative reactivity towards different functional groups in the same molecule or different molecules
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What are the "diagnostic" AO inhibitors?
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Menadione, Raloxifene, Vanillin, Hydralazine
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What are the 2 forms of Molybdenum Hydroxylases
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Xanthine oxidase and aldehyde oxidase
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what are the 2 proposed paths for oxygen insert on a hydrocarbon from compound I? |
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What are the absorbance peaks for oxidized, reduced, and ligand bound P450
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oxidized - 412 nmreduced - 417 nmligand bound - 450 nm |
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what determines whether a FMO can oxidize a soft nucleophile
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Accessibility, FMO will oxidize just about any soft nucleophile the FAD-OOH encounters |
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what do microsomal and mitochondrial p450's specialize in?
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Drugs and xenobiotics // steroids and other endogenous compounds
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What do XO and AO target?
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SP2 carbons that are electron deficient because of adjacent nitrogens or oxygens
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what does b5 do?
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It donates and accepts single electrons (for the second reduction step). It's thought to increase coupling by increasing then decreasing the oxidation rates.
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What does FMO stand for?
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Flavin Containing Mono Oxygenases
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What does it mean that the FMO reaction mechanism is ordered?
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The cofactors and substrate all add to the enzyme before anything leaves
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What form of the P450 complex gives the characteristic 450 nm absorbance?
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Fe(2+) -CO(Bound to Carbon Monoxide)
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What happens to KIEs when the distance between sites (on the molecule) is supressed?
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The KIE ratio is "masked" / suppressed
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What is "metobolic switching" in reference to KIEs?
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when a P450 switches to a different site preference upon deuteration
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What is a good FMO inhibitor for diagnostic purposes
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there isn't a good chemical inhibitorFMO's are vulnerable to heat, so a slight raise in temperature can effectively inhibit them
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What is a Reverse Type I substrate?
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It displaces the H20 ligand, but the the heme stays in a low spin because of the 6th associated Fe ligand (can still be metabolized)
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What is a True Intermediate
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True Intermediates in a chemical reaction always give the same products, regardless of how the intermediate was produced
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What is a Type I substrate?
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it displaces the H20 ligand, converts a low spin complex to high spin complex, and doesn't associate with the heme |
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What is a Type II substrate?
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Displaces H20 and has a strong field ligand functional group that is usually nitrogen |
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what is stereoselectivity
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selection among several substrates or from several products
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What is stereospecificity?
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The retention, inversion or loss of stereoselectivity. A reaction is stereospecific if stereoisomeric substrates give specific stereoisomeric products
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What is the (general) ease of hydroxylation, not counting orientation in the active site? |
tertiary (benzyllic and allylic) > secondary > primary |
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What is the AO substrate probe? |
Pthalazine |
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What is the classical endogenous FMO substrate? |
Frimethylamine (TMA)
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what is the difference between high spin and low spin |
high spin - out of plane (usually unbound)low spin - in plane (usually bound to something)
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What is the FAD catalytic cycle? |
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What is the flow of electrons in Molybdenm Hydrolase Complexes
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From substrates to electron acceptors (usually oxygen)
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What is the FMO posthetic group?
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FAD-OOH It's weak compared to the Heme |
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What is the general AO activity dogma?
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it's high in monkeys and humans, low in rodents, and absent in dogs
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What is the input of protons controlled by?
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The proton transfer groove of the I helix: {ASTG}{ASTG}{DEQN}T{ASTG}
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What is the major form of FMO in human livers? |
FMO3- adultFMO1- fetal
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What is the p450 signature sequence?
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FxxGxxxCxG
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What is the rating of FMO selectivity?
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FMO1 (least selective) -> FMO2 -> FMO3 (most selective) |
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What is thought to be the purpose of the proximal cysteine?
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It regulates the activity of the heme
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Whate are the homology rates for families and subfamilies of p450? |
40% and 55% respectively
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Where do the reductive electrons for p450s come from? |
NADPH -> CPR -> p450
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Where does FMO oxidize
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soft heteroatom nucleophiles (N,S,P,Se)
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Where is FMO1 found in adults
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the kidney |
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where is the heme center in a p450 structurally?
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buried beneath the I and L helices
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Which cofactor is REQUIRED?
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CPR (cytochrome P450 Reductase)
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Why is FMO2 generally not considered in drug studies? |
polymorphisms (Q472X) render it inactive in all but a small population
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what are the diagnostic MAO inhibitors |
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What does CES stand for and what does it do |
Carboxylesterases hydrolize esters |
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What does EPHX stand for and what does it do? |
Epoxide Hydrolase, hydrolyses epoxides |
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What structural class of enzymes do CES and EPHX belong to? |
alpha/beta hydrolase folds |
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What is a catalytic triad? |
three amino acids that cooperate to actiate a water molecule and form OH- and H+, then use those ions in a chemical reaction |
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Why are CES especially important from a clinical viewpoint? |
Ester derivatives are often used as prodrugs to improve absorption, bioavailability, taste, stability, and duration of action |
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How many CES genes are there? |
at least 5: 1,2,3,4A, and 5A |
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What is the CES catalytic triad? |
Ser (Glu/Asp), His |
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What is the carboxylesterase catalytic cycle? |
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How do A and B esterases interact with organophosphates |
A esterases hydrolyze organophosphates B esterases are irreversibly inhibited by organophosphates |
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What does PON1 Stand for |
Paraoxonase |
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What does PON1 do? |
It is a lactonase and metabolizes phosphoric triesters, which can be toxic organophosphates |
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What type of compound often is in insectisides? What bioactivates them? |
Organophosphates (OP) that are actiated by P450 into oxons (toxic) |
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How are oxons toxic |
They bind irreversibly to acetylcholinesterase |
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How does PON1 neutralize oxon toxicity? |
It hydrolyzes the oxon: |
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What is the PON Mechanism? |
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Where are carboxylesterases found? (CES) |
they're errywhere, but concentrated in the small intestine, lung, and liver |
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What enzyme metabolizes methyl-ester cocaine? |
CES1 |
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What enzyme metabolizes Meperidine |
CES1 (slowly) |
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What enzyme metabolizes delapril |
CES1 |
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What enzyme metabolizes 4-methylumelliferyl-acetate? |
Mostly CES2 (60,000) with some CES1(2000) |
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What enzyme metabolizes the benzoyl ester cocaine? |
CES2 |
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What enzyme metabolizes heroin? |
Mostly CES2, with some CES1 |
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What enzyme metabolizes 6acetyl morphine? |
Mostly CES2 with very very little CES1 |
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What enzyme metabolizes CPT11 |
Mostly CES2 with a little CES1 |
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what effect does esterase activity have on clopidogrel efficacy? |
It drastically decreases the amount of active metabolite by hydroxylating the ester. Clopidogrel is a prodrug |
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Why does prasugrel generate more active metabolite than clopidogrel? |
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Which CES isoforms are in the liver |
BOTH! (but more CES1) |
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Genetic CES1 polymorphisms have drastic effects on what drug? |
Methylpheidate (Ritalin) |
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How were BChe Polymorphisms discovered? |
Paralysis medications lasted much longer than anticipated (patients required assisted breathing apparati) |
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What does BChE stand for? |
Butyryl-cholinestease |
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What is the dominant drug metabolizing enzyme of the eye? |
BChE |
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where is BChE found? |
Ubiquitously, but we focus on plasma levels in metabolism |
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What do epoxide hydrolases typically do? |
they catalyze the formation of vicinal diols from epoxides |
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Which epoxide hydrolase is soluable and which is microsomal |
EH1 - microsomal |
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where is microsomal epoxide hydrolase found? |
primarily in the liver but can be found in most tissues |
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What are in vitro mEH probes |
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What is the mEH mechanism of epoxide hydrolysis? |
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what does EH do with stereochemistry |
It inverts it |
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What does valproic acid do with mEH |
it inhibits it and can lead to Drug Drug Interactions |
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What are the phisiological roles of sEH and why do we care |
aracadonic, lineolic, and other fatty acid epoxides are metabolized by it, these are relevant because they regulate blood pressure and inflammation |
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What is the general structure of the sEH inhibitors developed by Bruce Hammock? |
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What is the proposed catalytic mechanism of sEH? |
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What is the most common metabolic reduction that drugs, xenobiotics, and endogenous compounds undergo? |
Reduction of carbonyls |
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What does ADH stand for |
Alchohol dehydrogenase |
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What does CR stand for |
Carbonyl Reductase |
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What is the ADH cofactor? |
NADH, it is the only reductive nzyme that uses NADH, all the others use NADPH |
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Draw the single electron reduction of quinones |
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what is futile cycling |
when a cycle restarts itself, it can be a problem because it wastes resources without any useful production |
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How can quinones cause toxicity |
they react with thiols to form protein adducts, and react with molecular oxygen to create Reactive Oxygen Species (ROS) in a futile cycling process |
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Why is NQO reduction of quinones less toxic than other methods |
it's a 2 electron process that bypasses the radical forms of quinones |
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What does NQO stand for? |
naptho-quinone oxido-reductase |
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Why is NQO1 relevant to chemotherapy |
It's upregulated in some cancers? |
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Where are azo and nitro reactions typically catalyzed |
Intestinal microflora |
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Why is analine a structural alert |
oxidation by p450's results in very reactive species |
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Why is reductive or oxidative dehalogenation of CCl4 very bad? |
Reductive - results in carbon radicals that lead to lipid peroxidation Oxidative - generates trifuuroacetylaldehyde - which leads to immune hepatitis |
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What do N-acetyl-transferases (NAT) catalyze? |
the transfer of an acetyl group from acetyl-CoA to primary arylamines and hydrazine |
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What is the exception to "xenobiotics containing primary amines are rarely subtrates for N-acetyltion?" |
Cysteine conjugates, which are formed from glutatione conjugates and are converted to mercaptic acids by N-acetylation in the kidney |
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Where is NAT1 expressed? |
errywhere |
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What does NAT1 acetylate? |
"monomorphic substrates" Ex: sulfamethiazole p-aminosalysilic acid |
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Where is NAT2 expressed |
primarily liver and intestinal mucosa |
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What does NAT2 acetylate? |
"polymorphic substrates" sulfamethiazine, isonazid, dapsone, sulfamethoxole, procainamide, hydralazine, and caffeine |
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What drug first identified slow acetylators |
isonazid |
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What is the primary cause of slow acetylators |
NAT2 polymorphism |
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What is "wild type" NAT2 responsible for fast acetylation? |
NAT2*4 |
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Diagram the role of NAT in amine genotoxicity |
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What is the methyl donor for S-methyl transferase, O-methyl-transferase, and N-methyl-transferase? |
S-adenosyl-methionine (SAM) |
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There are at least 2 distinct S-methyl transferase enzymes, what are they and what do they require? |
TMT- thiol methyl transferase |
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What is COMT |
catechol o-methyl transferase |
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What does COMT metabolize |
mainly neurotransmitters |
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Where does COMT methylate |
The meta position
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What can decrease UDPGA stores in humans? |
Starvation and Alchoholism |
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What does UDPGA stand for? |
Uradine diphosphate glucuronic acid |
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How does UDPGA work? |
it creates a good leaving group, then a SN2 reaction with a nucleophile results in a glucuronide |
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How do CYP metabolism and glucuronidation relate? |
Every time a CYP metabolizes a compound, it's a glucuronidation target |
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how do C and S glucuronidation compare to O and N glucuronidation? |
C and S are much more rare |
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What makes a site good for glucuronidation |
electron density/nucleophilicity |
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Where are UGT's found |
the luminal side of the endoplasmic reticulum (CYPs are on the cytosolic side) |
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How specific are UGTs? |
While there are some examples of specificty, not all reactions of UGT are specific |
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What does UGT stand for? |
UDP glucuronosyl Transferase |
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What iss thought of UGT dimerization? |
UGT can function as a dimer but might not have to be a dimer to function (it's not completely known) |
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What domain is completely conserved across all UGT isoforms |
The cofactor binding domain |
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Why can glucuronides result in analytical difficulties for drugs like statins or NSAIDs, |
futile cycling/ regeneration of aglycone |
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What interesting thing can happen with acyl glucuronides |
acyl migration |
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What can happen to nucleophiles (like Cs) with Glucuronides |
they can Acylated which can then be immunogenic |
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Why do we not freak out about protein conjugates of glucuronated drugs? |
only a small percentage actually gets there |
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What is the difference between a diglucuronide and a bis-glucuronide |
diglucuronide - linked glucuronide bis-glucuronide - two distinct sites of glucuronidation |
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What is PAPS |
3'phospho adenoside - 5 phosphosulfate |
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what's the difference between cytosolic and Golgi SULTS? |
Cytosolic: metabolize small molecules |
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How does sulfonation compare to glucuronidations substrate specificity |
It's a narrower range: only soft nucleophiles, no thiols or carbons |
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What 3 peptides are glutatione formed from? What's the special linkage? |
Glutamate, cystein, and glycine. The amide bond between glutamate and cysteine is with the glutamate's side chain |
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What is interesting about the Glutathione cysteine? |
It's redox active |
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What type of drugs react with glutathione? |
Electrophillic drugs |
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What is the "universal" GSH substrate |
1-chloro-2,4,dinitrobenzene CDNB |
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When adding glutathione to an aryl amine, what drives dehydratin |
Rearomatization |
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What are the 2 structurally unrelated forms of GST? |
MAPEG - membrane associated eiconasanoid and glutathione metabolism |
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What is the functional form of a MAPEG |
trimers |
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Where are MAPEGs found? |
the endoplasmic reticulum |
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What is the functional form of cytosolic GSTs |
Dimers |
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What is the purpose of the GST "mu" loop |
it allows for diverse substrates |
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What is interesting about GSH reactions with Isocyantes and isothiocyantes |
GSH addition is reversible and can accidentally transport the drug/toxin |
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Why is APAP toxic |
Futile cycling can lead to depletion of Glutatione stores (NAPQI) |
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What does glutathione do to isomerize double bonds |
It acts as a base, binds, then leaves (allowing the bond to isomerize) |
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Describe the interaction between GSTs and GSH |
it is a weak but specific binding, it's acceptable because there's so much GSH |
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What form of glutathion binds to GST? |
GS - |
