Enzyme Kinetics Flashcards
What are the proportions of reactants and products when Kc > 1?
Products exceed reactants
- much greater than 1, reaction almost at completion
What are the proportions of reactants and products when Kc < 1?
Reactants exceed products
What factor affects Kc?
Temperature
What factors do not affect Kc?
- pressure
- catalyst
Zeroth law of thermodynamics
If 2 thermodynamic systems are in thermal equilibrium with a 3rd, then they’re in thermal equilibrium with each other.
First law of thermodynamics
Energy can’t be created/destroyed - can only be transferred.
Second law of thermodynamics
The entropy of an isolated system not in equilibrium will tend to increase over time, approaching a maximum value at equilibrium.
Third law of thermodynamics
As temperature approaches absolute zero, the entropy of a system approaches a constant minimum.
Define enthalpy ΔH
The change in heat associated with a chemical reaction.
What reactions take place when ΔH<0 or ΔH>0?
- Spontaneous exothermic reactions ΔH<0 (negative)
- Non-spontaneous endothermic reactions ΔH>0 (positive)
Define entropy ΔS
The degree of disorder
- Matter changes from a more ordered to a less ordered state.
(randomness in a system)
What reactions take place when ΔS<0 or ΔS>0?
- Spontaneous reaction ΔS>0 (positive)
- Not spontaneous reaction ΔS<0 (negative)
Gibbs Free Energy equation
ΔG° = -RTln(Kc)
What does the Gibbs Free Energy equation calculate?
Measures the difference in free energy between the products and reactants of a reaction.
- it is a thermodynamic property
(determines whether reaction is spontaneous or non-spontaneous)
What is the relationship between ΔG and standard-state to equilibrium?
- The smaller the value of ΔG, the closer the standard-state is to equilibrium.
- The larger the value of ΔG, the further the reaction has to go to reach equilibrium.
When do exergonic reactions occur?
- Spontaneously
- ΔG is negative
reactants E > products E
Draw an energy profile for an endergonic reaction
Slide 16 (part 1)
What factors affect reaction rate?
- temperature
- concentration
- addition of catalyst
- SA
Under what conditions can a reaction spontaneously occur?
- thermodynamically favourable
- kinetically favourable
What is the Collision Theory?
Reactions occur when molecules collide with:
- sufficient energy
- correct orientation
So bonds can be broken/made
How can the rate of a reaction be increased on a energy profile?
Increase number of successful collisions:
- change shape of curve
- move activation energy further to the left
What factors affect reaction rate explained by collision theory?
- increase in temp
- increase in conc of reactants
- addition of catalyst
How does the collision theory explain temperature as a factor?
Increase temp, rate of reaction increases.
- molecules move faster
- collide more often
- increase in number of high energy collisions
How does the collision theory explain concentration as a factor?
- higher concentration of reactants
- chances of collision are greater
How does the collision theory explain catalysis as a factor?
Increases rate by increasing number of successful collisions.
- remains unchanged whilst providing lower activation energy
Does a catalyst change equilibrium state?
No - ΔG° and Kc remain the same.
What are enzymes?
Proteins that act as catalysts - biological catalysts that increases reaction rate.
How do enzymes function?
- temporarily binding
- specifically activating a substrate
What are the 6 types of enzymes (we need to familiarise with)?
- oxidoreductases
- transferases
- hydrolases
- lyases
- isomerases
- ligases
What type of reaction do oxidoreductases catalyse?
oxidation-reduction reactions
What type of reaction do transferases catalyse?
transfer of functional groups
What type of reaction do hydrolases catalyse?
hydrolysis reactions
What type of reaction do lyases catalyse?
group elimination to form double bonds
What type of reaction do isomerases catalyse?
isomerization
What type of reaction do ligases catalyse?
bond formation coupled with ATP hydrolysis
What is a kinase (enzyme)?
An enzyme that catalyses the transfer of a phosphate group from ATP to a specified molecule.
What is the enzyme-substrate equation?
E + S ⇌ ES
What is the 1st step at the active site of an enzyme?
S binds to active site to form ES
What is the 2nd step at the active site of an enzyme?
As S binds to active site:
- enzyme adjusts its conformation
- so active site & S take the shape of the transition state
What is the 3rd step at the active site of an enzyme?
The transition state-enzyme complex gives rise to product.
What is the 4th step at the active site of an enzyme?
- product released from active site
- enzyme is returned unchanged to its native conformation
- ready to bind to another S
What are the characteristics of enzymes?
- Increase speed of reaction: 10^6 - 10^14 rate enhancements
- very selective
- very specific
= responds to regulation
How much do enzymes increase rate of reaction?
Factors of 10^7-14
What is meant by Enhancement Factors?
Enhancement Factors refer to various strategies or factors that can increase the rate or efficiency of a chemical reaction.
What are the enhancement factors in enzyme catalysis?
- distortion of substrate
- provision of functional group
- provision of microenvironment in active site
- controlling the orientation of reactants
- reducing unfavourable interactions between reactants in multi-substrate reactions
- proximity effect
How does distortion of the substrate affect the rate of a chemical reaction?
- distortion of the substrate towards the shape & electronic structure of the transition state
- increases the rate of a chemical reaction by lowering the Ea required for the reaction to occur
How can the provision of a functional group affect the rate of a chemical reaction?
- provision of a functional group for an alternative reaction mechanism
- increases the rate of a chemical reaction by providing an alternative, lower-energy pathway for the reaction to occur
How can the microenvironment in the active site of an enzyme affect the rate of a chemical reaction?
- provision of a microenvironment in the active site - affects the rate of a chemical reaction
- by favouring a particular reaction pathway
- through factors such as pH, ionic strength, and electrostatic interactions
How can controlling the orientation of reactants affect the rate of a chemical reaction?
- controlling the orientation of reactants can increase the probability of a chemical reaction occurring
- by bringing the reactive groups into closer proximity
- & minimizing non-reactive interactions
How can Enhancement Factors reduce unfavourable interactions between reactants in multi-substrate reactions?
- reduce unfavourable interactions between reactants in multi-substrate reactions
- by stabilizing the transition state
- & minimizing repulsion between reactants
What is the proximity effect in relation to Enhancement Factors?
- proximity effect increases probability of a chemical reaction occurring
- by bringing the reactants into close proximity
- thereby increasing the likelihood of productive collisions
How can the average rate of reaction be measured?
Measure change in reactant/product over a given time period.
- slope between two points
How is the instantaneous rate of a reaction found?
Particular point - tangent of slope
What is the equation for rate of reaction?
rate = k[A]^x
What is k in the rate of reaction equation?
k = rate constant
What is ^x in the rate of reaction equation?
the order of reaction with respect to a particular reactant
What is the overall order of reaction?
Add the powers
What is a zero order reaction?
x = 0 so
rate = k[A]^0
rate = k x 1
rate = k
What is the half-life of a reaction (t1/2)?
Time taken for concentration of reactant to fall to 50%?
How does half-life decrease in a zero order reaction?
Half life decreases as concentration of reactant decreases
- so half-life dependent on concentration
What is the relationship between half-life and concentration in a first order reaction?
Half-life is independent of concentration of reactant
- half-life remains constant.
What is V0?
Initial velocity / rate of reaction
What is measured instead when [ES] is difficult to measure?
Measure V0:
- with fixed amount of enzyme
- and excess substrate
- when product is at a minimum
What graph is the rate of an enzyme reaction shown on?
[P] against time graph
How is initial velocity (V0) measured on a graph?
When [S] is a maximum at:
- time = 0
- and [P] is at minimum
What happens to V0 when [E] is constant and [S] increases?
- [E] constant
- [S] increased
- V0 increases
What curve is shown on a V0 vs [S] graph?
- hyperbolic curve
- approaches a maximum value of V0
- approaches Vmax asymptomatically
(part 2, slide 22)
What is Vmax?
Maximum rate/velocity of the enzymatic reaction where all enzyme is bound to substrate (saturated with substrate).
- theoretical maximum rate of reaction
- never achieved in reality
- units = μmol / mL / s
How is Vmax approached on a V0 against [S] graph?
Asymptomatically approached as [S] is increased.
What is the Michaelis-Menten Equation?
Graph resulting from plot of V0 against [S]
What is the Michaelis Constant (Km)?
[S] at 1/2 Vmax
- half of enzyme is bound to substrate
- units = moles L-1
What is Km used to evaluate about an enzyme?
The specificity of an enzyme for its substrate.
What else does Km measure of an enzyme?
Measures affinity of the enzyme for the substrate.
What does a high Km suggest?
Weak association between E and S
- high Km = low affinity
What does a small Km suggest?
Enzyme binds strongly to its substrate
- low Km = high affinity
- Enzyme works at low [S]
How are the characterisations of enzymes determined?
By measuring these parameters:
- Vmax
- Km
- turnover number (kcat)
Define turnover number (kcat)
Measure of catalytic activity
- maximal number of S molecules
- catalysed per unit of time
- by a S saturated enzyme
What is the Lineweaver-Burke plot?
Straight line graph
- 1/[S] (x) against 1/V0 (y)
- X-intercept = -1/Km
- Y-intercept = 1/Vmax
- m = Km/Vmax
What is the Lineweaver-Burke equation?
1/V0 = (Km/Vmax)1/[S] + 1/Vmax
What is a con of a Lineweaver-Burke plot?
Puts unneeded emphasis on the data corresponding to small values of [S].
What is a Hanes-Woolf plot?
Straight line graph
- [S]/v (y) against [S] (x)
Why is the Hanes-Woolf plot used more frequently than the Lineweaver-Burke plot?
- gives more reliable values of Km and Vmax
- when experimental methods prone to errors
- error distribution better in H-W than L-B
What are the limitations of Michaelis-Menten
- only applicable to single substrate & single product reactions, so does not allow reversibility of product formation, P & S inhibition
- assumes that the reaction is irreversible
- assumes that enzyme concentration is much smaller than substrate concentration
- does not account for enzyme cooperativity (where the binding of 1 S molecule affects the binding of subsequent S molecules)
