Reaction Kinetics and Inhibition Flashcards
What is the order of a reaction?
It tells you how much the concentration of a reactant affects the rate
Order corresponds to the molecularity of the reaction = the number of molecules that must simultaneously collide to generate a product
What is the individual order? Overall order? Rate equation?
Individual order - the power to which a concentration is raised in the rate equation
Overall order - the sum of the individual orders in the rate equation
Rate equation - rate = k[A][B]
k = rate constant
What is first order? Second order?
First - the rate is proportional to the concentration
Second - the rate is proportional to the square of the concentration
What was the first proposed equation?
E + S ⇌ ES ⇌ P + E
K1 - rate over first arrow
K2 - rate over second arrow
K-1 - rate coming back over first arrow
K2 is the rate determining step (slowest)
How was the Michaelis-Meuten equation derived?
To derive the equation you have to make an assumption and the equation will differ slightly based on which assumption you assume
There were 2 possible assumptions:
- Equilibrium assumption - E + S and ES are in equilibrium
- Steady state assumption - ES maintains a steady state, as [S] > [E], until [S] is exhausted
Steady State was used
What is the Michaelis-Menten equation?
Vo = Vmax[S] / Km + [S]
What is the Mechaelis constant?
Km = K-1 + K2 / K1
What is Vmax?
The maximal velocity/rate of a reaction, when the enzyme is completely saturated with its substrate in the ES form
What is Km?
Substrate concentration that gives you 1/2 Vmax
What does Km tells us?
Km tells us something about the affinity of the enzyme for the substrate:
Low Km = high affinity
High Km = low affinity
What kind of graph is produced when plotting [S] against Vo?
Rectangular Hyperbola
It has an asymptote
What is a measure of catalytic efficiency?
kcat/Km = specificity constant
Which is a useful indicator of relative catalytic efficiency
This ratio can’t be bigger than K1, most values are near the diffusion controlled limit of 10^8 to 10^9 M-1 s-1
What is kcat?
The turnover number - the number of reaction processes that each active site catalyses per unit of time
kcat = Vmax/[E]T
[E]T = total enzyme conc
What can be used to measure Km and Vmax?
A Lineweaver-Burke Plot
y = mx + c
1/Vo = Km/Vmax * 1/[S] + 1/Vmax
How to draw a lineweaver-burke plot?
Plot 1/V on the y axis
Plot 1/[S] on the x axis
What can we determine from a lineweaver-burke plot?
Y-intercept = 1/Vmax X-intercept = 1/Km
Slope = Km/Vmax
What is used to provide the information for a lineweaver-burke plot?
Assays - used to measure how much an enzyme there is
Continuous - e.g. measuring absorbance in a spectrophotometer
Discontinuous - stopping the reaction and measuring
Coupled - measuring another reaction coupled to the reaction we want to measure
Quantified by taking the initial rate of reaction (tangent to a curve)
What is the problem with steady state kinetics?
It doesn’t reveal the number of intermediates in the enzyme cataylsed reaction
Therefore intermediates have to be identified through the use of spectroscopic techniques
Knowledge of intermediates is essential to confirm/eliminate proposed mechanisms
What are bisubstrate reactions?
Reactions with multiple substrates yeilding multiple products
e.g.
Sequential reactions occur via single displacements
AND
Ping pong reactions occur via double displacements
What are some types of sequential reactions?
Ordered mechanism - complusary order of the substrate addition to the enzyme
Random mechanism - both binding sites are present on the free enzyme (any order)
What are ping pong reactions?
Group-transfer reactions in which one or more products are released before all substrates have been added
The substrates don’t encounter each other on the surface of the enzyme
What are some factors affecting enzymes?
Temperature
pH
Substrate concentration
Enzyme concentration
Inhibitor presence/concentration
Describe the effect of temperature on enzymes?
Non-covalent interactions can break and the protein can denature - disrupting the active site
Thermophilic enzymes can survive above 100 degree
Our enzymes are mesophilic
Psychrophilic enzymes - exist in freezing temperatures
What do inhibitors do? Types?
They decrease enzyme activity
Irreversible - covalently binds so tightly to the enzyme it permenantly blocks enzyme activity
Reversible - diminishes activity but can be removed
Types of reversible inhibitors?
Competitive - competes with the substrate for the binding site on the enzyme
Uncompetitive - Inhibitor binds to the ES complex
Non-competitive (mixed) - Can bind to both the E or the ES complex
Describe competitive inhibition? Examples?
The inhibitor binds to the enzyme forming an EI complex
E.g. malonate is a competitive inhibitor for succinate going to fumarate
AND
using transition state analogs to inhibit the affects of HIV/AIDs
What equations can work out Ki for competitive inhibition?
Vo = Vmax[S]/ aKm + [S]
a = 1 + [I]/Ki
a = apparent
How does competitive inhibition affect Vmax and Km?
Vmax - unchanged
Km - increases
Describe uncompetitive inhibition?
Inhibitor binds to the ES complex forming an ESI complex
What equations can work out Ki for uncompetitive inhibition?
Vo = Vmax[S]/ Km + a’[S]
a’ = 1 + [I]/K’i
How does uncompetitive inhibition affect Vmax and Km?
Vmax - Decreases
Km - Decreases
Describe noncompetitive inhibition?
Can bind to both the E or the ES complex, forming either EI complex or a ESI complex
What equations can work out Ki for noncompetitive inhibition?
Vo = Vmax[S]/ aKm + a’[S]
a = 1 + [I]/Ki a' = 1 + [I]/K'i
How does noncompetitive inhibition affect Vmax and Km?
Vmax - Decreases
Km - Increase or decrease
What is pure noncompetitive inhibition?
Only Vmax is affected and aKm remains unchanged
Said to be similar to irreversible inhibition
