Enzymes 2 Flashcards
Enzymes are proteins that speed up (catalyse) specific chemical reactions. They have various functions which include (5)
- Digestion: Carbohydrates, fats, proteins
- Blood clotting: fibrin clot catalysed by thrombin
- Defence-immune system activation of complement
- Movement: Muscle actomyosin is an ATPase
- Nerve conduction: Membrane ion pumps for Na+ and Ca2+
Key enzyme properties include: (5)
- Increase reaction rate
- Show specificity
- Unchanged at end of reaction
- Do not alter reaction equilibrium
- Facilitate reaction by decreasing the free energy of activation of the reaction
Explaining further on from that last property of enzymes they work by decreasing the free energy of reaction
what is needed for a reaction to be favourale?
what is the highest point of free energy of activation?
We know that in a chemical reaction where there is conversion of reactant to product, it involves a change in the free energy of the reaction. – progress of the reaction.
Whenever there is a drop in free energy from reactant to product, the reaction is favourable, but to get to the product you have to put free energy in first which is like a barrier.
This amount of energy is the ‘free energy of activation’.
The highest point of the free energy of activation is the transition state.
Enzymes speed up reactions by reducing the free energy of activation
the enzyme uses the binding energy of its substrate to lower the activation energy
We also categorise enzymes according to their kinetic parameters, there are two very important parameters which are the: (2)
- Vmax = maximum rate of enzyme reaction
* Km = A measure of how tightly the substrate is able to bind to the enzyme active site
Vmax
what is it?
• Vmax is when working flat out, every active site at every moment is doing something.
o So, the Vmax is a measure of the ES complex -> E+Product.
Km
what is it? what does it tell us?
The Km is the substrate concentration at which the Vmax is half. This tells us how sticky the substrate is for the enzyme. (affinity)
Enzyme Catalysis
Are enzymes perfect? Where they have the maximum catalytic activity.
what does enzyme bind to? what does this form? what 2 things can happen from here?
what are the 2 broad characteristics of enzym reaction?
what can evolution work on from these 2?
so how do you work out if an enzyme is perfect or not?
Enzyme molecule binds substrate to form enzyme-substrate complex.
The ES can do two things:
- Either break up and form back into the E+S
- The chemistry can kick in and we get the enzyme and product.
Hence, we can think of enzyme reaction as having two broad characteristics
• First enzyme has to bind substrate, and this is limited by diffusion
• The second step is all the chemistry on the active site
So, we think about which part of this can evolution work on? The answer is evolution can only really work on the chemistry of the enzymes -> speed them up.
Therefore, one way of knowing if an enzyme is perfect is that it is not limited by its chemical activity, so a perfect enzyme reaction rate is only limited by diffusion.
‘Perfect’ enzymes- reaction rates limited by diffusion. Evidence?
3 things?
1.Is the reaction rate affected by viscosity? Does a change in viscosity by adding glycerol slow the enzyme reaction rate? (hence diffusion affected)
2.Theoretical calculation for diffusion limited reaction-
k3 /Km ~ 108 M-1s-1?
Carbonic anhydrase: k3 is ~600,000 s-1 ; Km is 8 x 10-3
3.Determine complete free energy profile for the enzyme reaction. Is substrate binding rate limiting?
We can calculate this from modelling what the diffusion limited rate would be:
K3 / K,m = 108 M-1s-1
what is the chemical rate constant? what do we divide it by?
what does this measure the rate?
what must the ratio be about?
how do you get K3?
what is an example of a perfect enzyme?
- K3 is the chemical rate constant and we divide it by Km.
- This measures the rate from E+S all the way to E+P, if the ratio is about 108M-1s-1 then you have a diffusion limited reaction. Many enzymes have this but not all.
- We can get K3 by doing Vmax/[enz]total
Carbonic anhydrase = perfect enzyme, an example of a diffusion-limited enzyme.
So why has evolution not made all the other enzymes “perfect”?
why? (2)
The answer is that it wouldn’t be helpful to have all the enzymes working full out all the time, as all the available nutrients would be degraded.
- Enzyme activity needs to be controlled e.g. allosteric, phosphorylation etc.
- Some enzymes have to sacrifice their efficiency in order to be controlled.
TIM
Another perfect enzyme is one in glycolysis, this enzyme is called TIM (triosephosphate isomerase).
what does this enzyme do? from what?
how does the enzyme do this?
what is the rate limiting step?
This enzyme interconverts two products.
On breakdown of fructose 1,6 bisphosphate you get two products, but only one is useful which is glyceraldehyde-3-phosphate.
- The other molecule has to be converted to G3P, TIM catalyses this reaction.
- The enzyme does this by carrying out lots of small steps rather than one large step. The smaller steps all have lower activation energies.
- The chemical steps have the lowest activation energies compared to others. This shows how nature has made is so that the chemical reaction is not the limiting step.
o The intermediates have roughly the same free energy too.
o The rate limiting step would be the release of the products.
Proteases
what do they do?
A protease is an enzyme that breaks down other proteins, they hydrolyse the peptide bonds of their protein substrate.
Proteases are of interest as they are therapeutic targets. We know there are various classes of proteases such as: serine-, cysteine, aspartyl and metallo proteinases. All which hydrolyse peptide bonds
We will focus on serine proteases as they have a very reactive serine at their active site.
Serine proteases
what are two involved in digestion?
what is involved in lung function?
why are all serine proteases very reactive?
what else makes it very reactive?
We have many serine proteases in our body, two involved in digestion are chymotrypsin and trypsin. There is also elastin which is important in lung function.
These enzymes are very similar in their structure and how they work.
All the serine proteases are very reactive because they have something called the catalytic triad, there are other residues nearby that are able to H bond with the serine and the histidine present also helps make the serine more reactive by moving a proton.
Chymotrypsin
what does it do? what does this form?
Chymotrypsin has a very reactive serine which attacks the peptide bond by forming an acyl-enzyme intermediate. (which is very easily hydrolysed by water)
