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What is the function of a spectrophotometer?
Measure the amount of light that passes throught the cuvette
Which wavelength is optimal to measure NADH?
Define the absorbance value displayed on the spectrophotometer.
It is the logarithmic ratio between light falling on the sample and passing through it, log(l0/lt)
What is included in the master mix?
Ethanol, NAD+, and buffer
Why don't we add the enzyme into the master mix?
The reaction will start immediately, and our measurement will be inaccurate.
What role does ethanol play in this reaction?
Substrate for the enzyme
What is the function of NAD+ in this reaction?
Why do we need buffer in the master mix?
To stabilize reaction pH
What is the substrate concentration in this master mix?
What aspect of a reaction do they [enzymes] lower, thereby increasing the reaction rate dramatically?
What is the function of the NAD+ cofactor?
It acts as an electron transporter or electron acceptor
What was just shown in the animation?
The enzyme-substrate complex was formed
When the reaction produces no intermediates, which of the following rate constants will be equal to kcat?
Calculate the ethanol volume for two master mixes with final substrate concentrations of 0 mM and 0.0053 mM in a final volume of 1 mL.
0 mL and 0.106 mL
Which part of the curve should you use to measure the intial reaction rate?
What is the optimal enzyme concentration?
What is the optimal pH for the alcohol dehydrogenase enzyme?
What is the optimal temperature for the alcohol dehydrogenase enzyme?
How can we convert this into product concentration?
A = e • c • l
What values do we need to calculate to plot Michaelis-Menten curve?
Initial reaction rate
What is the estimated Vmax for wild type ADH?
What is the estimated Vmax for mutant ADH?
Looking at the plot, which one is Km?
What is the Km for wild type ADH?
What would the Km be for the mutant ADH?
What would explain the difference observed in the two enzymes?
A base change in the DNA
When analyzing inhibition kinetics, which plot is useful in determining the type of inhibition taking place?
What kind of inhibitor is Hydroxyalime?
In competitive inhibition, how are the Vmax(app) and the Km(app) affected?
What is the Vmax(app) value for the Hydroxylamine inhibition?
How can you calculate the Ki for a competitive inhibition?
By comparing the slopes of Lineweaver-Burk plots at different inhibitor concentration
What is the Ki value for Hydroxylamine?
What kind of inhibitor is Cimetidine?
In noncompetitive inhibition, the Vmax(app) is ... and the Km(app) is ...
What is the apparent Vmax value at 500 μM cimetidine?
What is the Ki value for the Cimetidine inhibition?
What kind of inhibitor is Formamide?
In uncompetitive inhibition, the Vmax is ... and the Km is ...
What is the Km(app) value for the 100 μM Formamide inhibition?
What is the Ki value for the Formamide inhibition?
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