MCDB 310: CHAPTER 6
32 Cards in this Set
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Enzymes (what are they, differ from Hb +Mb, structure, function, requires)
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Usually proteins (except small RNAs--> ribozymes)
Differ from Hb and Mb by not only binding ligand but by changing chemical structure (of the substrate)
Specific 3d-structure --> specific active site (catalytic center) --> catalyze a specific chemical reaction
often requires cofac…
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Terms associated with enzymes
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cofactor
coenzyme
prosthetic
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Cofactor
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non-protein component (e.g. metal ions) of enzyme required for activity
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Coenzyme
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Cofactor is complex organic molecule (e.g. vitamin)
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Prosthetic group
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Coenzyme is tightly or covalenty bound (e.g. heme in cytochrome c oxidase)
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Terminology for enzyme needing cofactor
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Apoenzyme (incative -usually protein part) + cofactor (non protein component) --> Holoenzyme (active)
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Enzymes as catalysts (vs. peptide synthesis vs chemical catalysis, degree of activity)
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Can accelerate chemical reactions 10*5-10^17 times
much greater specificity than chemical rxns (e.g. peptide synthesis)
uses much milder rnx conditions than chemical catalysis
degree of activity can be regulated
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What exactly do enzymes establish?
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Increase the RATE of a rxn without changing the free energy change or the equilibrium
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Why do we need activation energy?
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Energy required in chemical reactions to:
align reacting groups
form temporarily unstable charges
rearrange bonds
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Enzyme effect on activation energy
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Enzyme decreases activation energy ΔG++ and with that increase the rate of the rxn
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How do enzymes increase the reaction rate?
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provide favorable microenvironment for reaction e.g. by:
aligining the reacting groups
stabilizing charges
rearranging bonds
ultimately stabilizing the transition state
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Enzymes and organizing reactive groups (how, also called)
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Enzymes organize reacting groups into close proximity and proper orientation
at active site, both reacting groups are held as if in one molecule
also called"increase the local concentration
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How to lower activation energy
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Enzymes bind the transition state better than they bind the substrate or product
Active site assumes a transition state complementing comformation:
1. substrate binds w/ a subset of interactions to the active site
2. substrate undergoes conformational changes to assume transition state…
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Transition state stability
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Transition states, by definition are too unstable to be observed or captured
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Can we develop antibidoes that bind the transition state and use them as enzymes?
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Sort of
Trick is to use transition state analogs (stable molecules that closely mimic transition state structure as antigens)
antibodies made against transition state analogs, bind to transition state and make it more stable e.g. cocaine
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What are the catalytic mechanisms?
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Acid base catalysis
Covalent catalysis
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Definition of kniematics
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study of rate at which compounds react
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Rate of enzymatic reaction is affected by
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enzyme
substrate
effectors
temperature
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How do we perform kinetic measurements?
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2. mix enzyme + subsrate
2. record rate of product formation as function of time (velcotiy of reacction
3. V0 is the velocity at time = 0
4. Change substrate concentration and determine V0
5. plot initial velocity V0 versus substrate concentration
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What are inhibitors?
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Compounds that decrease enzymes activity
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Reversible inhibitors do what? often used? Ways they bind?
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Bind to and can dissociate from the enzyme
-often used as structural analogs of substrates or products
often used as drugs to slow down a specific enzyme
can bind to free enzyme and preent binding of substrate
to enzyme substate complex and prevent the reaction
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competitive inhibition (binds to, acts, effective at what [S], Km or vmax affected)
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binds to same site as substrate and competes w/ it
acts by reducing free concentration of enzyme available for substrate binding
effective at low [S]
increasing [S] will outcompete inhibitor --> Km is HIGHER b/c more [S] is neede to overcome ihibition
Vmax is unchanged since s can …
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Competitive inhibition and Km, inhibitor dissociation constant
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KI = [E] * [I]/ [EI]
Km increases by factor of α, X intercept decreases by -1/αkm
α = 1 + [I]/KI
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Uncompetitive Inhibition (how it works, effective at what [S], inhibitor dissociation const
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Inhibitor binds to ES complex but not to free enzyme and affects the maximal activity of the enzyme
Does not need to resemble substrate
causes structural distortion of active site w/out substrate binding
inhibitor binding distorts the ES complex, which inhibits catalysis
most effect…
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Uncompetitive Inhibition and Vmax and Km
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Vmax is lowered
Because I stabilizes ES, less substrate is needed to reach saturation --> KM is lowered
Both Vmax and Km decrease by α'
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Mixed inhibition( how it works, Vmax and Km, effective at what [S]
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Inhibitor binds to both enzyme and ES complex
Vmax decreases by α'
Km altered by α/α'
Can work at high and low [S]
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Non-competitive inhibition
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A special case of mixed inhibition
When Ki = Ki' --> only Vmax reduced but Km unchaged
Inhibitor binds E and ES with same affinity
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Example for mixed inhibitors
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Ritonavir -HIV treatment
Inhibitor of HIV1 Protease
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Types of enzyme activity regulation, what does it affect
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Regulation can be:
-noncovalent modification
covalent modification
irreversible
reversible
affects mainly enzymes that catalyze reactions at break points of metabolic pathways
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Allosteric Enzyme (what are they, types)
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very similar to hemoglobin
Do not follow michaelis menten kinetics
usually multimeric proteins that are regulated by reversible, noncovalent binding of regulatory component (modulator)
homotropic modulator or heterotropic odulator (non subsrate) which either:
stimulate enzyme activi…
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Regulation by reversible covalent modification (phosphorylation example)
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Phosphorylation/Dephosphyorylation
Phosphorylation: Catalyzed by kinases
Dephosphorylation: Catalyzed by phophatases
Very important in signal transduction cascades
Phosphorylation causes conformational changes in enzyme, which usually activate enzyme
Phosphorylation sites: Hodroxyl…
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Activation by irreversible covalent modification (zymogens example)
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enzyme is actually made, transcribed, translated and folded in its inactive form, inactive form = zymogen
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MCDB 310: CHAPTER 2