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How do enzymes offer rate acceleration/enhancement?
By lowering activation energy (EA)
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Why did nature evolve enzyme? What do enzymes afford and how do they affect Gibbs Free Energy (ΔG)?
- Biological reactions cannot simply rely/depend on spontaneity, some reactions require huge EA, and would take an eternity to go to completion
- Enzymes afford reaction rate acceleration by lowering the barrier to activation (EA), they have NO EFFECT on Gibbs Free Energy (ΔG)
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Where do the rate enhancing reactions provided by enzymes take place?
Catalysis takes place in a region called active site
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Define active site and state the expected residues)
Region of an enzyme that contains catalytic residues (from CDESTY HKR category)
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Label both figures and state the expected ΔG (explain your choice)
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What is the difference between ΔG and the activation energy?
- The ΔG is a difference in a free energy of the products and reactants
- The activation energy is a measure of energy required to break covalent bonds in reactants
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What is the difference between an enzyme catalyzed reaction versus a non-catalyzed reaction?
A catalyzed reaction ensures LOWERING of the activation energy
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The apex or peak of the curve is where the ________ state lies, it is extremely short-lived and _______
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Define substrate
Substrate: a molecule upon which the enzyme acts. Enzymes catalyze chemical reactions involving the substrate(s). In the case of a single substrate, the substrate bonds with the enzyme active site, and an enzyme-substrate complex is formed.
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How does the rate of the reaction relate to the amount of substrate concentration, [S]?
- At low [S] (red circle), rate is directly proportional to [S]
- At higher [S], curve levels off and asymptomatically approaches the Vmax of the reaction
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Define Km
- Km (Michaelis constant): the substrate concentration at 1/2 the maximum velocity
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If Km = [S] at half the maximum velocity, then V0 = ____
In other words, what can Km be estimated as?
- 1/2Vmax
- estimated as 1/2Vmax
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When [S] = Km, exactly half of the ______ _____ have been filled with _______
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If Km value is large, enzyme binds substrate ______. This means there is a large(r) probability that enzyme-substrate complex can ________. If the Km value is small, enzyme binds substrate more ______; small(er) probability that enzyme-substrate complex will ______
- weakly
- dissociate
- strongly
- dissociate
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What is kcat? State the equation too
- The turnover number (kcat): The turnover number defines the number of substrate molecules formed into products per unit of time
- Kcat = Vmax/[E]
**[E]: Enzyme concentration
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What would be the kcat of an enzyme that has a Vmax of 60,000 M/s and an enzyme concentration of 0.1M?
(60,000 M/s)/0.1M = 600,000s
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How does kcat relate to the rate of the reaction?
- Higher the kcat, the FASTER the reaction
- Lower the kcat, the SLOWER the reaction
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The reciprocal of turnover number can provide us with the time it takes to?
Catalyze a single reaction
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Why evolve enzyme inhibtors? (2)
- Enzyme function needs to be modulated in the cell (NATURAL REGULATION)
- Enzyme function needs to be halted or blocked (DRUGS)
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Define Irreversible inhibition
Modification of the enzyme (active site) via covalent binding aka suicide inhibition
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Reversible inhibition
Modification of the enzyme (active site) via non-covalent binding
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What are the 4 types of reversible inhibition?
- Competitive
- Non-competitive
- Uncompetitive
- Mixed inhibition
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Define Competitive and Non-competitive inhibitors
Competitive inhibitors: inhibitor (similar structure to substrate) binds to active site reversibly; prevents binding of substrate. For example, Tamiflu binding to viral neuraminidase or Statins such as Lipitor bind to HMG-CoA reductase
Non-competitive inhibitors: binds to enzyme AWAY from active site; binding alters enzyme shape, thus affecting catalysis
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Define Uncompetitive and Mixed inhibition
- Uncompetive inhibition: binds ONLY to enzyme-substrate complex
- Mixed inhibition: inhibitor binds to enzyme in presence or absence of substrate. However, it should be noted that sometimes there is a preference
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Define Uncompetitive and Mixed inhibition
- Uncompetitive inhibition: binds ONLY to enzyme-substrate complex
- Mixed inhibition: inhibitor binds to enzyme in presence or absence of substrate. However, ti should be noted that sometimes there is a preference
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Generally explain what is happening in this reaction?
- This is irreversible inhibition, an example of the mechanism and mode of action of a common non-steroidal anti-inflammatory drug (NSAID)
- Aspirin acetylates a serine residue on cyclooxygenase (COX-1)
- Acetylserine is formed
- Leads to irreversible inhibition of enzyme
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How is the type of inhibition best determined?
Through use of what are known as double-reciprocal plots aka Lineweaver-Burk or a double-reciprocal plot of non-competitive inhibition
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What are 4 factors of non-compeitive inhibition
- Inhibitor doesn't interfere with the binding of substrate to active site since the inhibitor doesn't occupy same site
- Inhibitor (I) and Substrate (S) are different shapes
- Vmax is altered
- Binding to inhibitor alters enzyme shape affecting catalysis
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How to determine the Km value from plot?
Extrapolate line (use a ruler!), make best estimate of x-intercept, solve algebraically
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What does this plot tell you about the Km and Vmax?
- Km is unaffected
- Vmax is reduced
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Label the graph and state the type of inhibition. How are Km and Vmax affected? Can the inhibitors be catalyzed by the enzyme?
 - Km increased
- Vmax unaffected
- Inhibitors CANNOT be catalyzed by the enzyme
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What kind of inhibition is displayed? How are Km and Vmax affected?
- Mixed inhibition Lineweaver Burk
- Km increased (in the presence of inhibitor)
- Vmax decreased
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Depict a Lineweaver-Burk Plot for uncompetitive inhibition. How are Km and Vmax affected?
 - Km reduced (in presence of inhibition)
- Vmax decreased
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Where do uncompetitive inhibitors bind? Uncompetitive binding of inhibitor stimulates binding of _______! What does this cause?
- Bind to a site other than the active site
- substrate!
- Causes lowering of Km in the presence of inhibitor
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Fill out the table for each of the 4 types of inhibition
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Draw and describe the following situations:
No inhibition
Irreversible inhibition
Competitive inhibition
 - No inhibition: substrate binds normally
- Irreversible inhibition: imagine a deprotonated serine side chain in covalent engagement with the inhibitor, I.
- Competitive Inhibition: Inhibitor competes with normal substrate for binding to the active site
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Draw and describe the following situations:
Non-competitive inhibition
Uncompetitive Inhibition:
Mixed inhibition:
 - Non-competitive inhibition: Inhibitor binds to an allosteric site AWAY from enzyme altering enzyme's shape and function
- Uncompetitive inhibition: Inhibitor binds AWAY from active site. Binding of inhibitor stimulates enhanced binding of substrate lowering Km
- Mixed inhibition: Inhibitor can bind to enzyme alone AWAY from active site OR inhibitor can target the enzyme-substrate complex. In both cases, inhibitor binds AWAY
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We have been with the following data for an enzyme known as butyrylcholinesterase. Based on the double-reciprocal plot, please determine:
The type of inhibition
- Solution: Obtain reciprocal values and ALWAYS label and show units
- x-axis: 1/[S] (mM)-1
- y-axis: 1/V0 (min/mM)
 
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PT I: In the table below, you have been provided with enzyme kinetics data investigating the rate of the enzyme, ATP sulfurylase, in the presence and absencce of the inhibitor, chlorate
Find your x and y values and graph
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PT II: Determine Km and Vmax in both the presence and absence of an inhibitor
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