Enzymes Flashcards
give some examples of enzyme functions
o Digestion : carbohydrates, fats, proteins
o Blood clotting: fibrin clot catalysed by thrombin
o Defence-immune system-activation of complement
o Movement: muscle actomyosin is in ATPase
o Nerve conduction: membrane pumps for Na+, K+, Ca++
How do we usually categorise enzymes and give some examples
We usually categorise enzymes according to the particular type of reaction they catalyse eg: o Proteases( break down proteins), nucleases( nucleic acids), polymerases ( form polymers), Kinases ( transfer phosphate groups).
enzyme defects cause disease. give three examples of inherited diseases involving an amino acid, a sugar and a complex lipid.
o Phenylketonuria: patient lacks a liver enzyme which converts Phe to Tyr. Therefore, levels of phenylalanine, if left untreated rise in the body and the breakdown products can poison and impair the development of the brain. Treatment: phenylalanine deficient diet – the levels can be contolled
o Glycogen storage disease- lacks an enzyme which mobilizes glucose from glycogen. It cannot stabilise glucose
o Tay-sachs disease-defect which prevents processing of a membrane ganglioside-neuronal damage and death
give some examples of enzymes as drug targets.
o Antibiotics: eg penicillin inhibit cell wall synthesis
o Anti-inflammatory agents: aspirin blocks prostaglandin synthesis
o Anti-cancer drugs : methotrexate is a folate analogue : interferes with synthesis of DNA precursors required for DNA synthesis in cancer cells, blocking the replication of cancer cells.
outline the key properties of an enzyme
o Increase reaction rate by up to 10 billion fold.
o Show specificity
o Unchanged at end of reaction
o Do not alter equilibrium
o Facilitate reaction by decreasing the free energy of activation of the reaction.
How do enzymes speed up reactions by?
reducing delta G
what is the free energy of activation delta G dagger?
- The free energy of activation delta G is the difference in free energy between the substrate and the transition state.
describe how enzymes speed up reactions by reducing delta G
- Free energy of the substrate is higher than the free energy of the product. Therefore from substrate to product there is a release of free energy. This means that the reaction is thermodynamically favourable and can proceed.
- However, just because its thermodynamically favourable does not mean it will take place. This is because from substrate to product, energy is required to go into the system.
- In the uncatalyzed reaction, energy is required to push the system over the transition state (point of highest free energy) so that it can form the product. The free energy of activation is high
- In the catalysed reaction, the enzyme can reduce this energy barrier so that the free activation is lower and therefore we can put less energy into the system for the reaction to occur.
what is an active site
- The active site is a 3-D cavity or cleft that binds substrate(s) with specificity through electrostatic, hydrophobic, hydrogen bonding and van der Waals interactions.
- In the enzyme catalysed reaction, the Formation of an enzyme-substrate (ES) complex at the active site is the first step in enzyme catalysis. Not available in the uncatalyzed reaction
where does evidence for active sites come from?
- X-ray crystallography (usually done at synchrotrons-Diamond, Oxford) – used to determine the structures of enzymes and enzyme substrate complexes.
- Kinetic studies of enzyme activity (simpler and quicker than crystallography).
describe how structure of a protein is determined by x-ray crystallography
- Technique where you purify a large amount of your enzyme or protein of interest.
- You crystalise the protein and place the crystal in the path of a powerful x-ray beam
- The molecules in the crystal diffract the x-ray beam, and you can record the diffraction pattern, which consists of a series of spots in various positions and have varying intensities.
- Allows you to deduct the structure of the individual molecules in the crystal.
describe the structure of the human RAS protein and include how we were able to observe this.
- Ras is found in the inner face of the plasma membrane and acts as a switch between the membrane and the nucleus and signals the cell to grow.
- Its structure is that of a protein made out of Beta sheets and alpha helices. It has GTP bound to its active site.
- Therefore x ray crystallography enables us to see the structures of the enzyme substrate complexes.
what has studying the structures of enzymes enabled us to distinguish?
- Studying the structures of enzymes have enabled us to distinguish between the two alternative models of how a substrate binds to the active site on an enzyme
describe the lock and key theory
- The lock and key theory : the active site of the enzyme is directly complementary to the structure of the substrate and comes together to form the ES complex.
describe the induced fit theory
- The induced fit theory: the enzyme active site is not directly complementary to the structure of the substrate. As the substrate beings to bind to the active site, the enzyme changes its shape so that the active site does become complementary to that of the structure of the substrate.
give an example of a protein that undergoes induced fit binding
the induced fit binding of glucose to hexokinase
describe the binding of glucose to hexokinase
o Hexokinase is an enzyme involved in the early stages of glycolysis.
o The enzyme has a lobular structure
o Through x-ray crystallography, we can get the structure of the hexokinase enzyme alone, and we can also get the structure of the hexokinase-glucose ES complex. ( one of its substrates is glucose).So we can compare the two structures that we get from the x-ray crystallography. Ie the complex of the enzyme without the substrate bound to it, and the substrate after once it did.
When glucose binds to it, the two lobes undergo a conformational change and envelopes the glucose. This is evidence that the binding of glucose to hexokinase occurs by an induced fit mechanism.
How do enzymes speed up their reactions?
They use the enzyme substrate binding energy when making the enzyme substrate complex. They use this energy to reduce the free energy of activation.
describe in detail the mechanisms through which enzymes speed up the reaction by using the enzyme substrate binding energy
1) To bring molecules together in the active site.
2) By constraining substrate movement in a way that they are ideally placed to undergo a reaction.
2) A particular bond in the substrate has to be broken to make the product. So the enzyme strains particular bonds in the substrate making breakage easier in the transition state. Therefore it needs less free energy to reach the transition state when the bond is already strained.
3) Some reactions require the change in the positive and negative charges in the substrate as they are converted to product. An enzyme uses the binding energy to stabilise the positive and negative charges in the transition state.
4) The enzyme can envelope the substrate to exclude water from the active site- make reaction go faster e.g hexokinase
5) To provide an alternative reaction pathway of lower energy e.g., this can be achieved by the enzyme taking place directly in the chemistry of the reaction eg through the formation of the covalent enzyme-substrate intermediate.
6) Use cofactors: bring new chemistry to the active site with NAD(H), FAD(H2), metal ions such as Mg2+. Enzymes can bind cofactors to its active site in addition to the substrate, providing an alternative chemical pathway with a lower free energy of activation.
