Enzyme Kinetics
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
What is the substrate concentration in this master mix?
0.0016 mM
What is the optimal enzyme concentration?
0.1 mg/mL
What is the Km(app) value for the 100 μM Formamide inhibition?
1 mM
What would the Km be for the mutant ADH?
1.9 mM
What is the Ki value for the Formamide inhibition?
100 mM
What is the Km for wild type ADH?
16 μM
What is the estimated Vmax for wild type ADH?
18 μM/min
What is the apparent Vmax value at 500 μM cimetidine?
240 μM/min
What is the Ki value for Hydroxylamine?
30 μM
Which wavelength is optimal to measure NADH?
340 nm
What is the optimal temperature for the alcohol dehydrogenase enzyme?
44°C
What is the Vmax(app) value for the Hydroxylamine inhibition?
480 μM/min
What is the estimated Vmax for mutant ADH?
480 μM/min
What is the Ki value for the Cimetidine inhibition?
500 μM
What is the optimal pH for the alcohol dehydrogenase enzyme?
8.5
Looking at the plot, which one is Km?
A
Which part of the curve should you use to measure the intial reaction rate?
A
How can we convert this into product concentration?
A = e • c • l
What would explain the difference observed in the two enzymes?
A base change in the DNA
What aspect of a reaction do they [enzymes] lower, thereby increasing the reaction rate dramatically?
Activation energy
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 function of NAD+ in this reaction?
Cofactor
What kind of inhibitor is Hydroxyalime?
Competitive
In uncompetitive inhibition, the Vmax is ... and the Km is ...
Decreased; decreased
In noncompetitive inhibition, the Vmax(app) is ... and the Km(app) is ...
Decreased; unchanged
What is included in the master mix?
Ethanol, NAD+, and buffer
What values do we need to calculate to plot Michaelis-Menten curve?
Initial reaction rate
What is the function of the NAD+ cofactor?
It acts as an electron transporter or electron acceptor
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)
When analyzing inhibition kinetics, which plot is useful in determining the type of inhibition taking place?
Lineweaver-Burk
What is the function of a spectrophotometer?
Measure the amount of light that passes throught the cuvette
What kind of inhibitor is Cimetidine?
Noncompetitive
What role does ethanol play in this reaction?
Substrate for the enzyme
What was just shown in the animation?
The enzyme-substrate complex was formed
Why don't we add the enzyme into the master mix?
The reaction will start immediately, and our measurement will be inaccurate.
Why do we need buffer in the master mix?
To stabilize reaction pH
In competitive inhibition, how are the Vmax(app) and the Km(app) affected?
Unchanged; increased
What kind of inhibitor is Formamide?
Uncompetitive
When the reaction produces no intermediates, which of the following rate constants will be equal to kcat?
k2
