S2: Enzymes II

Question 1

Functions of enzymes

Answer 1

• Digestion: Carbohydrates, fat, proteins
• Blood clotting: Fibrin clot catalysed by thrombin
• Defence immune system activation of complement
• Movement actomyosin is an ATPase
• Nerve conduction: membrane ion pumps for Na+ and Ca2+

Question 2

What are enzymes?

Answer 2

Enzymes are proteins that speed up (catalyse) specific reactions

Question 3

Properties of enzymes

Answer 3

• Increase reaction rate
• Show specificity
• Unchanged at the end of reaction
• Does not alter reaction equilibrium
• Facilitate reaction by decreasing the free energy of activation of the reaction

Question 4

Describe how enzymes work by decreasing free energy of activation

What is the transition state?

Answer 4

In a chemical reaction there is conversion of reactant to product which involves a change in free energy of 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 use the binding energy of its substrate to lower the activation energy.

Question 5

What are the two important kinetic parameters we classify enzymes by?

Answer 5

Vmax = maximum rate of enzyme reaction (maximum velocity)

Km = A measure of how tightly the substrate is able to bind to the enzyme

Question 6

Describe an Michaelis-Menten curve

Answer 6

Different substrate concentrations [S] are plotted against reaction velocity (V)

Vmax is when working flat out, every active site at every moment is doing something. So the Vmax is a measure of the ES complex -> E+P.

The Km is the substrate concentration at which the Vmax is half where half the active sites are filled. This tells us how sticky the substrate is for the enzyme (affinity).

Question 7

Typical enzyme reaction

Answer 7

E + S ES —> E + P

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 or the chemistry can kick in and we get the enzyme and product E + P.

Question 8

What are the two characteristics of a typical enzyme reaction?

Answer 8

First enzyme has to bind substrate and this is limited by diffusion

The second step is all the chemistry on the active site

 

Question 9

What can evolution improve in a typical enzyme reaction?

Answer 9

Evolution can only really work on the chemistry of the enzymes, speed them up.

ES –> E + P

Question 10

What is a perfect enzyme reaction limited by?

Answer 10

One way of knowing if an enzyme is perfect is that it is not limited by its chemical activity (as so fast), so a perfect enzyme reaction rate is only limited by diffusion.

Question 11

Equation from modelling what the diffusion limited rate

Describe it

Answer 11

K3 / Km = 108 M-1s-1

K3 is the chemical rate constant and we divide it by Km. What this is measuring is 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

Question 12

Equation for K3

Answer 12

K3 is the chemical rate constant

K3 = Vmax/[enzyme]total

Question 13

Name a perfect enzyme

Answer 13

Carbonic anhydrase

Question 14

Why hasn’t evolution made all enzymes perfect?

Answer 14

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.

Question 15

Describe the perfect enzyme TIM (triosephosphate isomerase)

Answer 15

Another perfect enzyme is one in glycolysis, this enzyme is called TIM (triosephosphate isomerase). 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.

Question 16

What is a protease?

Answer 16

A protease is an enzyme that breaks down other proteins.

They hydrolyse the peptide bonds of their protein substrate.

Question 17

Name some classes of protease

Answer 17

• Serine
• Cysteine
• Aspartyl
• Metallo proteinases

Question 18

What are serine proteases?

Give examples

Answer 18

Proteases with a very reactive serine at their active site

• Chymotryosin (made in pancreas) and trypsin are involved in digestion
• Elastin in lung function.
  These enzymes are very similar in their structure and how they work

Question 19

Why are serine proteases very reactive?

Answer 19

They have a catalytic triad.
There are other residues nearby that are able to H bond with the serine and the histidine present which helps made the serine more reactive by moving a proton onto the aspartate group.

Question 20

Mechanism of proteases

Answer 20

Peptide bonds are thermodynamically unstable so the enzyme catalyses a favourable reaction by reducing the free energy of reaction.
If you are a protease you need to hydrolyse the peptide bonds on proteins, if there is not an enzyme present the reaction isn’t favoured.

Not every peptide bond is hydrolysed by every protease, proteases have specificity.
They only hydrolyse particular peptide bonds

The OH on serine attacks the peptide bond in proteins and the intermediate formed is then attacked by H2O (hydrolysed) and this further digests the proteins.

Question 21

How is trypsin specific?

Answer 21

The trypsin can only hydrolyse the peptide bond if the square side chain is positively charged, so a lysine or arginine. This is because in the binding pocket of trypsin there is a binding site for the Lys/Arg and it has a negatively charged residue. This allows enzyme to bind to the protein and cleave the peptide bond.

Question 22

How is chymotrypsin specific?

Answer 22

It wants the square side chain to be Phe, Trp or Tyr, so the binding pocket is hydrophobic.

Question 23

How is elastin specific?

Answer 23

Elastase wants a small residue, its binding pocket is very small so only small residues can get through and bind.

Question 24

Describe ATPase as a nano-machine

Answer 24

Enzymes in our mitochondria do this. The outer membrane is permeable to many substrates while the inner membrane is impermeable to many substrates including protons.

In oxidative phosphorylation, protons are pumped into intermembrane space and as inner membrane is impermeable to protons, you can set up a protein gradient. You are storing energy as well as there being a charge difference (a potential difference across the membrane).

On inner mitochondrial membrane, we have ATP synthase which acts as a motor. There are three active sites activated by a rotating spindle. We can drive this motor by protons entering down their concentration/proton gradient and as the motor spins, ATP is produced.

Question 25

What is topoisomerase II?

Answer 25

Topoisomerase II is also a nano-machine, it is a molecular clamp that unlinks tangled chromosomes. It uses ATP to work the clamp.