W5 - Enzyme Kinetics Flashcards
What is a holoenzyme or an apoenzyme?
Holoenzyme - enzyme with co-factor, Apoenzyme - enzyme without co-factor
Why does changing the activation energy not change the deltaG of the reaction?
The start and end energies remain the same
Are all enzymes proteins?
No e.g. catalytic RNA
How do enzymes lower the activation energy?
They stabilise the transition state
What enzyme catalyses the conversion between carbon dioxide and carbonic acid?
Carbonic anhydrase
Active sites are unique chemical microenvironments and are more likely hydrophobic, what 4 weak interaction occur at the AS?
Electrostatic, H-bonding, VdWs and hydrophobic
Does making or breaking a bond release/require energy?
Making - releases, Breaking - requires
Enzymes have no change on delta G and ???
Eq constant
What is the equation for rate of reaction?
Change in products over change in time
What is the units for rate?
Mol per sec (Ms-1) (Mol/L/s)
What 6 things can determine RoR?
Conc, catalysts, temp, pressure, pH, number of collisions
What is the rate equation for A + B –> P?
V = k [A][B]
What are the units of k, the rate constant?
M-1 s-1
Over time the RoR will go down, why?
The amount of substrate decreases as it’s converted to product
At equilibrium, what is equal?
The rate of the forward and backwards reactions
What is V0?
The initial (fastest) rate of reaction
At a fixed enzyme conc, what happens to the RoR when the [substrate] increases?
RoR increases
How to calculate V0?
Draw tangent on curve that goes through (0,0)
What is on the x and y axis of the Michaelis Menten curve?
x - substrate conc, y - reaction velocity
What graph/experiment do you need to do before making the M-M curve?
Conc of product against time at multiple different substrate concentration, calculate V0 for each (plotted on M-M curve)
What is the Vmax on the M-M curve?
Where the graph plateaus
What is the M-M formula?
V = Vmax ( [S] / ([S] + Km)) where Km is the Michaelis constant
What are the 4 things that give evidence for ES complexes?
Structural data from crystallography, spectroscopic data, electrophoretic crossing line and saturation effect
How does the electrophoretic crossling line experiment give evidence for the ES complex?
As the complex forms the molecule gets larger and doesn’t travel as far
What are the 2 reversible reactions of enzyme?
E+S forms ES, ES forms E + P
What are the rate constants for each reaction in enzyme action?
E+S –> ES has rate constant of K1 and the reverse rxn has K-1, ES –> E + P has rate constant of K2 and the reverse rxn has K-2
Why can we ignore K-2 in the initial rxn?
The reverse rxn is minimal
What is Vmax?
Theoretical maximum rate of reaction
What is the equation for Vmax?
Vmax = K2[ET] where [ET] is total enzyme conc
Describe what the M-M curve tells you?
The rate is initially proportional to [S] but it reaches a saturation value (Vmax)
At Vmax the ES –> E+P part of enzyme action determines how quickly the enzyme catalyses the reaction, what is the K2 also known as?
The Kcat
What do you call the enzymes turnover reaction?
Catalytic power
What 3 things does Km depend on?
pH, temp and ionic strength
Km is equal to the conc of substrate required for the reaction velocity to be ….
Half it’s maximum value
When [S] = Km, then V =
Vmax/2
What does the Km tell you about the M-M curve?
The smaller the Km value the steeper the gradient of initial Ror
What is the equation for Km?
Km = (K-1 + K2) / K1
If K2 < K-1, Km is equal to the…
Dissociation constant of the ES complex (Kp)
Km tells us the affinity of ES complex, If the Km is large/small, what does this tell us about the binding in the ES complex?
Large - weak binding, small - strong
What is the diffusion limit?
The maximum value for K2/Km (the catalytic efficiency constant)
What does the value of K2/Km have to be between to be diffusion limited?
10^8 and 10^9
How to find Km on a graph?
Find Vmax, then half Vmax and draw across then down to find Km
If glucokinase acts faster but has a lower affinity for substrate and hexokinase acts slower but has higher affinity for substrate what does this tell you?
Glucokinase works for storage better at higher glucose levels and hexokinase works in muscles better at lower lower levels
What does the Lineweaver -Burk plot, plot?
1/V0 against 1/[S]
What does the gradient, the y intercept and the x - intercept of the L-B plot tell you?
Gradient = Km/Vmax, y - intercept - 1/Vmax, x - intercept - (-1/Km)
If 1/Vmax goes up what happens to the Vmax?
It decreases
If Vmax gets lower and Km remains the same what does it tell us about the activity and affinity of the ES?
Activity is lower and affinity is the same
What enzymes do not follow the M-M kinetics?
Allosteric enzymes (produces sigmoidal graph instead)
