Enzymes 2 Flashcards
Define ‘Enzyme’
Enzyme – Proteins that speed up (catalyse) specific chemical reactions
Describe the functions and properties of enzymes
Functions:
• Digestion: carbohydrates, fats and proteins
• Blood clotting: fibrin clot catalysed by thrombin
• Defense immune system – activation of complement
• Movement: muscle actomyosin is an ATPase
• Nerve conduction – membrane ion pumps for Na2+ and Ca2+
Enzymes key properties:
• Increase rate of reaction by up to 10bn fold
• Shows specificity
• Unchanged at the end of the reaction
• Do not alter equilibrium reaction
• Facilitate reaction by decreasing free energy of activation of the reaction
Describe the concept of a perfect enzyme
• A ‘perfect’ enzyme is an enzyme where the chemistry it carries out at its active site is now so efficient that the limiting step is finding the substrate.
• Reaction rate is limited by diffusion.
• Speed at which enzyme finds the substrate is fixed by the structure of the enzyme. Rate Limited by this speed.
• EZ= intermediate
• 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.
• So, we can think of enzyme reaction as having two broad characteristics
o First enzyme has to bind substrate, and this is limited by diffusion
o 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.
• So, 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.
• We can calculate from modelling what the diffusion limited rate would be, and we get this by doing k3/Km = 108 M-1 s-1
• K3 (catalytic rate) 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.
• We can get K3 by doing Vmax/[end]total.
• Graph Y axis = Free energy.
• Carbonic anhydrase is an example of a perfect enzyme, an example of a diffusion-limited enzyme.
Why has evolution not made all enzymes perfect?
why has evolution not made all the other enzymes “perfect”?
• 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.
• 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.
Describe Protease enzymes
• 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.
• 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.
• Chymotrypsin has a very reactive serine which attacks the peptide bond by forming an acyl-enzyme intermediate.
• 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. Serine OH has high degree of electronegativity, so it is a very good nucleophile = very reactive.
• Catalytic triad: side chains make OH much more electronegative.
o OH made very negative, (serine reactive side chain).
o Interferes with C-N bond
o Ester intermediate (acyl enzyme).
o Water hydrolyses the ester intermediate.
o Peptide bond cleaved.
• 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 peptide bonds, this is shown below:
- 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.
- Chymotrypsin has a different specificity, it wants the square side chain to be Phe, Trp or Tyr, so the binding pocket is hydrophobic
- Elastase wants a small residue; its binding pocket is very small so only small residues can get through and bind.
- All this specificity means there is selectivity in reaction but still the same chemistry.
- Serine proteases have a conserved 3-D structure with a charge relay system, the different proteases can accommodate to different side chains.
Describe enzyme vs nanomachinery and ATP synthesis
- Enzymes in our mitochondria do this. The outer membrane is permeable to many substrates while the inner membrane is impermeable to many substrates Inc. protons.
- Inner membrane: electron transport chains = respiratory chain.
- Protons are generated and exported out of mitochondria.
- In oxidative phosphorylation, protons are pumped into IMS 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 and activated by a rotating spindle (proton driven). There are three active sites. We can drive this motor by protons, as the motor spins we produce ATP
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