BIOLCHEM 415: LECTURE 8
35 Cards in this Set
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Enzyme Inhibitors
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-An inhibitor is a molecule that interferes with catalysis
-importance for biology- immediate way to regulate activities of enzymes and therefore, pathways of metabolism, or synthesis of DNA
-Can affect Km and Vmax
-inhibitors can be reversible or irreversible
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Classes of reversible inhibitors
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-Competitive
-Uncompetitive
-Noncompetitive
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Irreversible inhibitors
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-include Suicide Inhibitors
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Competitive inhibitor
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-binds to active site and competes with binding of substrate
-reduces catalytic rate by effectively reducing the proportion of enzyme molecules with bound substrate
-less sites for substrate to bind
-relieved by increasing substrate concentration(reversible)
-NO EFFECT ON Vmax
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uncompetitive inhibition
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-inhibitor only binds to the enzyme-substrate complex
-less enzyme available to bind substrate
-NOT relieved by increasing substrate concentration because substrate can't get in
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noncompetitive inhibition
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-inhibitor and substrate can bind simultaneously at different binding sites
-reduces catalytic rate decreasing the number of active enzyme molecules
-NOT relieved by increasing substrate concentration
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how inhibitors affect kinetics
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-as before, measure velocity(rates) at different substrate concentrations
-BUT, repeat with increasing concentrations of inhibitor
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lineweaver burk plot
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-x intercept: -1/Km
-y intercept: 1/Vmax
-slope: Km/Vmax
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Competitive inhibitor kinetics
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-increases the apparent Km(presence of inhibitor) but not the Vmax
-it is harder to form ES complex, higher substrate concentration needed to reach Vmax/2
-for line weaver burk plot, since Km is larger with competitive inhibitor, x intercept(-1/Km) will be smaller or closer to zero and …
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Uncompetitive inhibitor kinetics
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-decreases the apparent Vmax and the Km
-increased [S] cannot overcome inhibition, less ES to bind to
-line weaver burke plot, since Km and Vmax are both smaller, the x and y intercepts will be greater since they are both denominators for those intercepts, but slope is the same
-Km dec…
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"apparent"
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-presence of inhibitor
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Noncompetitive inhibitors
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-decreases apparent Vmax but NOT the Km(since binding site is still the same)
-effectively decreases available enzyme concentration
-line weaver: x intercept stays the same since Km is not affected but y intercept increases since Vmax decreases
-Slope increases since Km stays the same …
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Comparing types of reversible inhibitors
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-Competitive-Km increase
-Uncompetitive- Km and Vmax decrease
-Noncompetitive: Km stays the same but Vmax decreases
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irreversible inhibitors
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-contain reactive groups
-modify the enzyme or cofactor by FORMING NEW COVALENT BONDS
-DO NOT dissociate from the enzyme(bc of new covalent bonds), unlike reversible inhibitors
-three types:
---Group specific reagents(target some general group)
---Affinity Labels(reactive substrate a…
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functional group specific reagents
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-React with side chains of specific amino acids
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affinity labels
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-looks like the substrate but it has a couple things different
-gets in the active site and modifies
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suicide inhibitors
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-Mechanism based inhibitors
-reactive intermediate generated by enzyme that inactivates the enzyme(intermediate only occurs after the reaction starts)
-ex) penicillin resembles D-Ala-D-Ala
----intermediate is resistant to hydrolysis or nucleophilic attack
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transition state analogs
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-note: enzymes like to bind transition states more than substrate
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important properties of effective drugs
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-easily administered to patients (small tablets)
-minimal toxicity and side effects
-survive in body long enough to reach target
-not modulate properties of molecules other than the targets
-be cleared within a reasonable period of time to diminish toxicity
-not all are enzyme inhibi…
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two approaches to drug discovery
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-A- Compound- physiological effect- molecular target
-B-molecular target-compound-physiological effect
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absorption, distribution, metabolism and excretion of drugs
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-they get into the bloodstream, then into the target compartment where they bind
-binding to wrong compartments
-equilibrium between free and bound
-some must be metabolized by liver to become active
-half life of a drug determines administration- determined by the rate that is elimin…
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albumin carries lipophilic drugs in the blood(serum)
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-transport drugs through the body via cardiovascular system
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metabolism of drugs alters their activity
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high drug levels can lead to toxicity
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HIV and antiretroviral drugs
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-HIV is a retrovirus(RNA based genome) that infects human immune cells(macrophages and helper T cells) causes AIDS
-antiretroviral drug targets:
---HIV reverse transcriptase- virus ss RNA to ss DNA
---HIV integrase- incorporates transcribed viral DNA into host genomic DNA
---HIV prote…
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rational design of drugs that inhibit HIV protease
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Penicillin as a suicide inhibitor
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-mechanism based inhibitor
-inhibits bacterial cell wall synthesis
-penicillin contains a thiazolidine ring fused to a B-lactam ring
-penicillin looks like D-Ala-D-Ala unit, forms penicilloyl-enzyme complex. This complex is resistant to hydrolysis or nucleophilic attack from amino grou…
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the enzyme that crosslinks peptidoglycan chains are called
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-transpeptidases, which acts like a protease to form a acyl-enzyme intermediate
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B-lactamase
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-an enzyme that confers resistance to B-lactam antibiotics(ex. penicillin) by hydrolyzing the B-lactam ring
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B-lactam ring
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-4 member ring with a N and carbonyl group
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clavulanic acid
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-β-lactamase inhibitor
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NSAIDs
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-non-steroidal anti-inflammatory drugs
-inhibit cyclooxygenase(COX), a membrane protein
-Cyclooxygenase(COX)- catalyzes the first step in prostaglandin synthesis by converting arachidonate to prostaglandin H2 in two steps
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ibuprofen inhibits COX
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-ibuprofen(NSAID) binds reversibly in the hydrophobic channel leading to the COX active site, blocking arachidonate binding
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COX enzymes
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Cyclooxygenase)(COX))catalyzes)the)first)step) in)prostaglandin)synthesis)by)conver6ng) arachidonate)to)prostaglandin)H2)in)two)steps.
Prostaglandins)have)many)func6ons,)such)as) causing)the)contrac6on)or)dila6on)of)blood) vessels,)regula6ng)inflamma6on,)and)inducing) fevers.
COX)is…
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Transpeptidase
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-forms the peptide cross-links between the rows and layers of peptidoglycan
-inhibited by B-lactams (ex. penicillin)
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BIOLCHEM 415: LECTURE 8