Enzyme Kinetics in Vitro Flashcards
What reaction is catalysed by ADH
ADH catalyses the reversible interconversion of ethanol and acetaldehyde:
Ethanol + NAD⁺ ⇌ Acetaldehyde + NADH
How does the role of ADH differ between yeast and mammalian liver
Yeast ADH: converts acetaldehyde to ethanol, regenerating NAD⁺ (important in anaerobic fermentation)
Mammalian liver ADH: converts ethanol to acetaldehyde, helping remove toxic alcohol
Why can both liver and liver ADH catAlyse either direction of the reaction but tend not to
The reaction is reversible, but kinetic properties and cellular conditions (e.g. substrate concentrations, pH) bias one direction over the other in vivo
What coenzyme is involved in the ADH reaction and what are its oxidised and reduced forms
The coenzyme is NAD⁺ (oxidised), which is reduced to NADH during ethanol oxidation
How is ADH measured in vitro
By using a spectrophotometer to monitor the increase in NADH absorbance at 340 nm indicating the conversion of ethanol to acetaldehyde
What is the extinction coefficient (ɛ) for NADH at 340 nm
6.22 × 10³ M⁻¹ cm⁻¹
What law relates absorbance to concentration
The Beer-Lambert Law
A = ɛcl
where:
A = absorbance
ɛ = extinction coefficient
c = concentration (M)
l = path length (cm)
What happens to the reaction rate when the substrate is in excess
The initial rate is linear before reaching equilibrium, making it ideal for calculating enzyme velocity
How do you calculate the concentration of NADH from absorbance at 340 nm
Use Beer-Lambert Law:
c = A / (ɛ × l)
For standard 1 cm path length, c = A / 6220
How do you calculate the reaction rate in µmol·mL⁻¹·min⁻¹
- Measure ΔA340 per minute
- Divide by extinction coefficient (6220) to get [NADH] formed per min (M·min⁻¹)
- Convert to µmol·mL⁻¹·min⁻¹
- Multiply by total volume to get µmol/min
- Divide by volume of enzyme used (0.05 mL) to express as µmol·mL⁻¹·min⁻¹ of enzyme
Why is it important to calculate initial velocity
Initial velocity reflects the maximum catalytic efficiency before product accumulation or substrate depletion affects the reaction
What is the Michaelis-Menten equation
v = (Vmax × [S]) / (Km + [S]),
where:
v = reaction velocity
[S] = substrate concentration
Vmax = maximum velocity
Km = substrate concentration at half Vmax
What does the MM curve look like
A hyperbolic (saturating) curve, showing rapid initial increase in rate that plateaus as substrate saturates the enzyme
How is Km estimated from a MM graph
Locate the substrate concentration at which the velocity is half of Vmax
What does a low Km value indicate
High affinity of the enzyme for the substrate
What does Vmax represent
The maximum reaction velocity achieved when the enzyme is fully saturated with substrate
How would enzyme kinetics differ between yeast and liver ADH
Likely different Km values and Vmax, reflecting their optimised roles in ethanol production (yeast) vs ethanol detoxification (liver)
What are some limitations of in vitro enzyme assays
Conditions differ from the cell:
Lack of regulatory proteins
Non-physiological pH or temperature
Substrate and cofactor levels may not reflect in vivo conditions
