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Questions and Answers
The Michaelis-Menten equation describes the relationship between the rate of an enzyme-catalyzed reaction and the ______ of the substrate.
The Michaelis-Menten equation describes the relationship between the rate of an enzyme-catalyzed reaction and the ______ of the substrate.
concentration
Which of the following equations represents the law of conservation of mass in the context of enzyme kinetics?
Which of the following equations represents the law of conservation of mass in the context of enzyme kinetics?
Knowing the KM value allows for adjusting assay conditions to optimize the reaction rate.
Knowing the KM value allows for adjusting assay conditions to optimize the reaction rate.
True (A)
What is the significance of the equation [E]0 = [E] + [ES]?
What is the significance of the equation [E]0 = [E] + [ES]?
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Match the following terms to their corresponding descriptions:
Match the following terms to their corresponding descriptions:
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The equation [E]0 = [E] + [ES] represents the law of conservation of mass.
The equation [E]0 = [E] + [ES] represents the law of conservation of mass.
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What is the significance of the equation [E]0 = [E] + [ES] in the context of enzyme kinetics?
What is the significance of the equation [E]0 = [E] + [ES] in the context of enzyme kinetics?
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Which of the following is NOT a benefit of knowing the KM value for an enzyme?
Which of the following is NOT a benefit of knowing the KM value for an enzyme?
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Flashcards
kcat
kcat
Turnover number; the maximum number of substrate molecules converted to product by an enzyme per unit time.
SpC
SpC
Specific activity; amount of product formed per unit time per amount of enzyme.
Continuous assay
Continuous assay
An assay method where the reaction progress is monitored continuously over time.
Discontinuous assay
Discontinuous assay
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Michaelis-Menten equation
Michaelis-Menten equation
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Enzyme units of activity
Enzyme units of activity
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Specific activity
Specific activity
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Law of conservation of mass
Law of conservation of mass
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[E]0, [E], [ES] relationship
[E]0, [E], [ES] relationship
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Substrate concentration ([S])
Substrate concentration ([S])
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Study Notes
Enzyme Purification and Assay
- Lecture 3 reviews Lecture 2
- kcat and SpC are discussed
- Continuous and discontinuous assays are covered
- Enzyme units of activity and specific activity are explained
Michaelis-Menten Kinetics Review
- Scientists Leonor Michaelis and Maud Menten developed the Michaelis-Menten equation
- The equation describes the relationship between enzyme activity and substrate concentration
- Enzyme-substrate binding forms an enzyme-substrate complex (ES)
- The initial velocity, V₀, is initially high, but then plateaus reaching maximum velocity (Vmax)
- Vmax = Vmax [S] / (Km + [S])
- The Km is the Michaelis constant, representing the substrate concentration at half the maximum velocity.
- Km = (k⁻¹ + k₂)/ k₁
- The reaction shows a hyperbolic shape in a plot of V₀ vs [S]
Turnover Number (TN, kcat)
- Turnover number (TN, kcat) refers to moles of substrate transformed into product per unit time, per mole of enzyme under optimal conditions
- Molecular activity is equal to TN per mole of enzyme
- Catalytic center activity is equal to TN per active site
- kcat = Vmax/[E]₀ = µmol (S→P) × min⁻¹ × mL⁻¹ / µmol enzyme × mL¹
- Typically kcat values fall in the range of 50 to 10⁷ min⁻¹
- kcat alone is not a reliable predictor of reaction rate
Specificity Constant (SpC)
- TN or kcat is the maximum number of moles of substrate converted to product per unit time per mole of enzyme
- kcat is a measure of how rapidly an enzyme operates once the active site is filled
- V₀ = kcat [S] / Km under low [S] conditions
- The ratio kcat/Km is known as the specificity constant (SpC)
- SpC compares the rate of enzyme-catalyzed reactions to rates of random substrate-enzyme diffusion
- A maximum value for the second-order rate constant between a protein and small molecule is ~ 10⁸ to 10⁹ M⁻¹s⁻¹
- SpC is a measure of how effectively an enzyme catalyzes a reaction in comparison to the rate at which the substrate and enzyme diffuse to each other.
Assay Types: Continuous Assay
- Reactions are monitored continuously using various techniques: spectrophotometric, fluorescence, stopped-flow instruments, plate readers, or other instruments
- Calorimetric and chemiluminescent assays are also used
- Initial part of the curve used for dP/dt calculation in continuous assay
- Enzyme concentration [E] must fall within the range where product formation varies linearly with substrate concentration
- Dead time, initial slope, and post-collection are important for data analysis
Assay Types: Discontinuous Assay
- Kinetic data is collected at fixed time points
- Radiometric assays measure radioactivity incorporation into substrates
- SDS-PAGE, HPLC, FPLC, and TLC are used for monitoring protein substrates/products
- The rate of [P] formation (or [S] disappearance) should be linear across the incubation time
Coupled Reactions
- Some reactions are difficult to assay because a readily measurable compound/property isn't produced
- Couple a difficult-to-assay reaction to another that yields a measurable compound and measure the measurable compound
- Conditions for E₂ activity must be compatible with E₁ to measure simultaneously
- For coupled reactions, E₂ must be highly concentrated to convert all B to C
- The rate-limiting step is the rate of action of the first enzyme (E₁)
- The relative amounts of E₁ and A depend on the nature of the assay
- If E₁ is measured, A should be high
- If A is measured, A should be low
Enzyme Units of Activity
- Enzyme solutions are often quantified in arbitrary units due to low concentrations and varying purity
- International Unit (IU) is a standard unit
- 1 IU is defined as the amount of enzyme that transforms 1 µmol of substrate per minute under specific conditions
- Specific activity is the number of enzyme units per mg of total protein
- Total activity = (units/mg protein) x (total mg protein)
Purification and Purity Assessment
- Enzymes are separated from other cell components using various techniques such as differential solubility precipitation, chromatography, and electrophoresis
- Precipitation methods use salting out with (NH₄)₂SO₄, isoelectric precipitation, or other techniques to separate
- Chromatography techniques involve gel filtration, ion-exchange, hydrophobic, and affinity chromatography (often using His, Flag, or GST tags now)
- Electrophoresis methods use native or denaturing electrophoresis
- Yield and purification factor are crucial in assessing enzyme purification effectiveness.
Determination of Specific Activity
- Measuring volume, protein content, and enzyme activity is required
- Calculate total protein: sample volume x protein concentration (mg/mL)
- Calculate total activity: sample volume x enzyme activity (units/mL)
- Specific activity = total activity / total protein (units/mg)
- Higher yield means better enzyme recovery
- Higher purification factor implies a purer product
Properties of Enzymes
- Key properties that need to be determined include molecular weight, quaternary structure/subunit structure, amino acid composition, amino acid sequence, and 3-dimensional structure (via X-ray crystallography/cryo-EM or NMR)
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Description
This quiz covers essential concepts in enzyme purification and activity assays, including kcat and specific activity. It also delves into the Michaelis-Menten kinetics, discussing enzyme-substrate interactions and important parameters like Vmax and Km. Prepare to test your understanding of enzyme behavior and kinetics in this comprehensive review.
