Lecture 14 - Enzyme kinetics I Flashcards
How do enzymes lower the activation energy?
How do enzymes lower the activation energy:
1. Enzyme binding of substrate results in a reduction in the degrees of freedom of movement/rotation
2. Enzymes distort their substrates towards the transition state
3. The sum of the multiple weak bonding energies upon interactions between individual residues at the active site and the substrates can offset/pay for the activation energy
Specificity is determined by interactions with amino acid side chains within the co-enzyme binding pocket
Why are enzyme kinetics useful?
Enzyme kinetics are useful because
* Simple rapid and cost-effective approach to understanding mechanisms of enzyme catalysed reactions
* Estimation of binding affinities of an enzyme for its substrates or inhibitors. Measurement of catalytic rate and enzyme efficiency
* Understanding of the dynamic-mechanistic properties of an enzymes is a prerequisite for the design of enzyme inhibitors or the development of diagnostic clinical assays.
Clinical assays that rely on a measurement of enzyme activity (Biomarkers)
○ Aspartate transaminase - Released after damage to cardiac muscle or the liver
○ Alanine aminotransferase - Liver disease, enzyme in hepatocytes
Why is a tractable assy critical for studying enzyme kinetics?
A tractable (easy to control) assay is crucial for studying enzyme kinetics effectively.
Characteristics of Standard Assays:
Initial Velocity Measurement:
Initial velocity is typically measured in “standard” assays.
Measurement is taken before 10% of the substrate is consumed to mitigate the effects of product feedback inhibition.
Necessary Components:
The assay must contain required co-factors and be appropriately buffered to maintain the correct and constant pH.
Methods of Rate Measurement:
Product Appearance or Substrate Disappearance:
Rate can be determined by monitoring the appearance of products or the disappearance of substrates.
Measurement methods include spectrophotometry or separation techniques like chromatography.
Coupled Assays:
If direct measurement of substrates or products is not feasible, a coupled assay is used.
The product of the reaction under study becomes a substrate for another reaction, allowing for indirect measurement.
Example: ATP + Glucose → G-6-P + ADP (hexokinase reaction)
Coupled Reaction: G-6-P + NADP → 6-phosphogluconate + NADPH
Measure NADPH appearance at 340 nm.
What are steady state kinetics?
In order to express kinetic data as easily measurable quantities, steady state kinetics requires two assumptions about what happens when enzyme an substrate are mixed.
1. Assumption of equilibrium
2. Assumption of steady state - Steady state is the situation in which the rate of formation is balanced with the rate of destruction.
For a reaction obeying Michaelis-Menten kinetics, a plot of initial reaction velocity (V0) against increasing substrate concentration gives a rectangular hyperbola that is described by the Michaelis-Menten equation.
Michaelis-Menten equation - the rate of an enzyme-catalysed reaction is proportional to the amount of the enzyme substrate complex.
Define - Rate limiting step.
Rate-limiting steps (1902 Adrian Brown):
When [substrate] saturates and therefore entirely converts E to ES form, the second step of the reaction becomes rate-limiting and is now insensitive to changes in [substrate]
What are te parameters of michaelis-menten kinetics?
The Michaelis-Menten equation
Vo=(V max[s])/(Km+[s] )
KM = The substrate concentration at which the reaction velocity is half-maximal
Vmax = The maximal reaction velocity (varies with temperature and pH)
A low KM means there is a high affinity
A high KM means there is a low affinity
In the absence of using computer software, then taking the reciprocal of Michaelis-Menten equation allows a straight line graph to be drawn as otherwise we can only get a rough estimate. This yields the double reciprocal or Lineweaver-Burk plot.
On the Lineweaver-Burk plot:
X intercept = 1/Vmax
Y intercept = -1/KM
Slope = KM/Vmax
The catalytic constant Kcat = Vmax/[E]
Kcat/KM then provides the measure of efficiency. How likely if a substrate comes into contact with the enzyme it will be catalysed. The diffusion controlled limit for the frequency with which enzyme and substrate molecules collide in solution is in the range of 108-109 M-1s-1
Some enzymes have reached catalytic perfection which means that any time they collide with a substrate molecule the product forms.
“Circe effects” are an explanation for catalytic perfection. The Circe effect is the utilisation by enzymes of strong attractive force to lure a substrate into a site in which it undergoes an extraordinary transformation.
How is kinetic data used to distinguish between modes of inhibition?
Competitive inhibition
When the inhibitor and substrate compete for the active site
As more substrate is added the inhibitor is out competed. The Vmax stays the same however the Km will decrease as more substrate is needed to meet the Vmax.
Uncompetitive inhibition
This is a rare form of inhibition where the inhibitor binds to the ES complex. In this case both the Km and Vmax change. (parallel lines on Lineweaver-burk plot)
The Vmax changes as the [ES] available for conversion to product is reduced. They do this by changing the shape of the active site so the reaction can’t be completed.
Non-competitive inhibition
The inhibitor binds the enzyme irrespective of whether substrate is bound or not.
Vmax changes and Km stays the same.
How do we measure multiple substrate enzyme kinetics?
Multiple substrate reactions are categorised by the number of products and reactants. (uni, bi, ter, quad)
E.g. a reaction that formed two produces from two substrates would be bi-bi.
For bi-bi reactions there are three possible mechanisms. Ordered, random and Ping-Pong
Ordered bi-bi
The substrates bind sequentially to form a tertiary complex. The products are then formed and leave sequentially in the order they bound.
Random bi-bi
The reaction can take any route of pathway meaning that the binding and release of substrates and products is random/doesn’t matter.
Ping-pong
Substrate A binds and forms the product which is released and the enzyme forms a modified state (F-state) where it can then bind B and release the product reforming the normal enzyme structure so more of substrate A can bind (E-state).
E.g. Complex II succinate dehydrogenase
The heterotrimeric complex II in the E-state FAD is in the oxidised state
When succinate binds It loses two H+ to the FAD forming fumarate and reducing the FAD to FADH2. This means that the enzyme has been modified to the F state
The H2 are then used to convert UQ to UQH2 and FAD is oxidised so the E-state has been reformed.
Lineweaver-Burk plots can be used to differentiate bi-substrate mechanisms.
Double reciprocal plots for a Ping-pong Bi-Bi mechanism
By plotting 1/V0 against 1/[A] at various different concentrations of B. The plots for each different concentrations will be parallel.
Double reciprocal plots for a sequential Bi-Bi mechanism
The plots are similar but the Vmax changes
