Practical D Flashcards
What 5 properties do enzymes differ in?
- optimum pH
- Vmax
- Km (affinity for substrate)
- Temperature dependence and thermal denaturing
- Sensitivity to inhibitor
How were each of the 5 prpoerties changed in the practical?
- pH: 1-14
- Time: 0-60mins
- Enzyme concentration: 0-250 micromolar
- Incubation tempurature: 0-100 degrees
- Substrate concentration 0-250 millimolar
What is the optimum pH of an enzyme?
The pH at which the enzyme is at its maximum acticity
How does pH affect the formation of the enzyme-substrate complex?
BInding is dependent on the complementary of side chains of amino acids on the substrate and active site of enzyme
Changes in pH alter the ionisation of these side chains affecting binding
pH can also change the tertiary structure of the active site
What is the relative range of pH activity for chargd and uncharged side chains?
Uncharged chains have a broader pH range compared to charged chains i.e they are more sensitive the changes to in pH
How are enzymes, whose optimum pH differs from the intracellur pH, function?
Subcellular compartmentation allows the enzymes to function at its own optimum pH
At what pH should enzyme activity be determined?
Enzyme activity must be determined at the optimum pH
Descibe the relationship between incubation time and product formed?
The longer the incubation time, the more product form.
The relationship is not linear but hyperbolic.
Initial rate is far grater
What happens to all enzymes of time?
All enzymes denature over time
Why must enzyme activity be measured using the intial rate?
Even if the [Substrate] saturates the enzyme, most of it will be consumed at the start of the reaction so less substrate will be available decreasing teh rate of product formed.
Also as the reaction progresses, the reverse reaction increases
How do you determine the intial rate from the graph?
Plot the rate of product formed over the first few seconds.
Draw a tangent to the steepest part of the curve
What is the error associated with a short incubation time?
The concentration of the product at the start is very low so it difficult to measure the amount of product precisely resulting in analytical errors
What is the best incubation time?
A duration which allows for accurate measuring of the product ( insignificant analytical errors).
Duration is not too long that the curve begins to slope
What happens to enzymes at high concentrations?
Enzymes may aggregate or form dimers affecting the activity
What can happen to an enzyme at very low concentrations ?
Enzymes that are natural dimers/oligomers may dissociate changing enzyme activity
What can be the problem with low concentrations of an enzyme. What is done to overcome this problem?
If an enxyme is unstable at low concs, it may readily denature lowering enzyme activity. Inert enzyme is added to act as chaperone for purified enzyme
Why might a [enzyme],[substrate] graph not pass through the origin?
The product is being produce from an alternative pathway which does not involve the investigated enzyme