12 - Enzyme Kinetics I Flashcards
What does it mean to understand enzyme kinetics qualitatively?
How enzymes serve as catalysts and have specificity
What does it mean to understand enzyme kinetics quantitatively?
How reaction rates change with mechanisms, mutants, substrates, etc.
How do enzymes work?
They decrease energy to make product
What do enzymes do?
Enhance reaction rate under physiological temperature, pressure, and pH
In a reaction coordinate diagram, what parts are based on kinetics?
The peaks, and differences in energy between valleys and peaks
What determines the rate of a reaction?
The transition state
In a reaction coordinate diagram, what represents thermodynamics?
The difference in energy between the valleys (equilibrium)
What is the transition state?
The highest energy species on the reaction coordinate
In a multistep reaction, what determines the rate of the reaction?
The biggest energy gap (bottleneck)
What is the difference between a transition state and an intermediate?
Transition states have partial bonds and cannot be isolated, while intermediates have full bonds and can be isolated
What is an example of a transition state?
An Sn2 reaction with partial bonds with the leaving group and nucleophile
What is an example of an intermediate?
A planar carbocation from an Sn1 reaction
What types of molecules bond very tightly to enzymes?
Molecules that mimic the transition state
What is activation energy?
Energy required to form the transition state during a collision between reactants
What does the activation energy do?
It controls the rate of the reaction
How are metabolics coupled in a cell?
Through rates (kinetics)
What is the rate of a reaction?
How fast reactants / products change over time (dC/dt)
What is the Arrhenius equation?
k = A exp(-Ea/RT) (relate k to Ea)
In the Arrhenius equation, what is A?
Frequency factor (how long collisions occur, proper geometry needed)
What is a rate law?
An expression that reveals the effect of reactant concentrations on the reaction (at a constant temperature)
What is rate proportional to?
Frequency of collisions (concentrations)
How are the order of the reaction determined?
Experimentally
For A –> P, what is the rate law?
dP/dt = kA (dA/dt = -kA)
What is the relationship between A and P at t = 0, and t = n?
At t = 0, [A0] = [A]
At t = n, [A0] = [A] + [P]
(conservation of mass)
What are the units of rate?
M/s
For a 0th order reaction, what are the units of k?
M/s
For a 1st order reaction, what are the units of k?
1/s
For a 2nd order reaction, what are the units of k?
1/(Ms)
What is the rate law for a second order reaction (in terms of product)?
dP/dt = k[A][B] = k(A0-P)(B0-P)
How can a second order reaction be simplified?
If B0»_space; A0, then B0»_space; P
What is the simplified second order reaction?
P = A0(1-exp(-kB0t)) (pseudo 1st order)
What is the rate law for a reversible reaction?
dP/dt = k1(A0-P) - k-1(P)
In a multistep reaction, what is the rate limiting/determining step?
The slowest rate (the only rate you need to consider)
How does an active site assist in a reaction?
Acid/base catalysis, nucleophilic catalysis, increasing effective concentration, stabilize intermediates/transition states
How does an enzyme increase effective concentration?
Bring reactants close together
What is the general formula for enzyme catalysis?
E + S ES –> EP
What is the Michaelis complex?
The ES complex
What is measured from a P vs t graph?
Initial velocity (v0)
Why is initial velocity measured?
The rate changes over time (less S interacts), and it is linear initially
What is the shape of the P vs t curve?
Hyperbolic (saturation)
What is the shape of the v0 vs. S curve?
Hyperbolic (saturation)
At low S, how does V0 vary?
It is 1st order linear (more S is more v0)
At high S, how does v0 vary?
It does not change (enzyme is saturated)
What is the rapid equilibrium assumption?
- E and S are in rapid equilibrium with ES (k-1»_space; k2)
2. E «_space;S, so Sf = S
What is Ks?
Dissociation constant of ES
What is the formula for Ks?
Ks = [Ef][Sf]/[ES]
What is ES under the rapid equilibrium assumption?
[ES] = [E][S] / (Ks + [S]) ([Ef] = [E] - [ES], [Sf] = [S])
What is kcat?
k2 (rate of catalysis from ES –> E + P)
What is the rate of the enzyme equation under therapid equilibrium assumption?
v0 = k2[ES] = kcat([E][S]/(Ks + [S]))
What is the formula for vmax?
vmax = kcat[E]
What is the steady state assumption?
ES reaches a constant value soon after the enzyme is mixed (d[ES]/dt = 0, d[E]/dt = 0)
What formula is derived from the steady state approximation?
Synthesis = degradation k1[Ef][Sf] = (k-1 + k2)[ES]
What is the formula for [ES] under the steady state assumption?
[ES] = [Ef][Sf] / Km
What is Km?
Michaelis Menten constant
What is the formula for Km?
Km = (k-1 + k2)/k1
What is the Michaelis Menten equation?
v0 = kcat[E][S] / (Km + [S]) (note vmax = kcat[E])
In a vo vs. S curve, where is Km?
At v0 = vmax/2, Km = [S]
Which is more generally applicable: rapid equilibrium or steady state assumption?
Steady state assumption
What are the units of Ks?
M
What are the units of Km?
M
What are the units of kcat?
1/s
What happens if k-1»_space; k2?
Km = Ks (k-1/k1)
What does Kd represent (in terms of enzyme catalysis)?
A system with no catalysis (only dissociation)
What is Km a measure of?
Substrate binding affinity
What is kcat a measure of?
Turnover number of enzyme, number of chemical steps
What is the formula for enzyme efficiency?
kcat/Km
What are the units of kcat/Km?
1/(Ms) (same as 2nd order reaction rate)
What does kcat/Km represent?
Free energy differences between free E and S, and transition state
What experiment can be done to determine the effect of an H-bond on catalysis?
- Measure kcat/Km for wild type and mutant enzyme
2. Calculate delta delta G (-RT ln(ratio of kcat/Km for both))
What assumptions are present for seeing if an H-bond is important for catalysis?
- Is H-bond only thing changing?
2. Any changes in partial structure or folded structure?
