1.4 - Enzymes Flashcards
What are enzymes?
They are biological catalysts which mean their function is to control the rate of reaction without being used up
What type of protein is an enzyme?
Globular Protein
What is the structure of a globular protien?
They are made up of amino acids, most enzymes contain 100-1000 amino acids. The polypeptide folds up to produce a unique 3D structure –> secondary + tertiary structures
What is specificity?
Characteristics of enzymes that mean that as a result of their very specific shape from their tertiary/quaternary structures, each enzyme will only catalyse a specific reaction or group of reactions.
So enzymes show a great specificity.
Lock and Key Hypothesis
- only a particular substrate or substrates with a particular structure will fit in the active site of an enzyme. Giving enzymes high specificity
- active sites affect the bonds in a substrate and brings them close together, bonds can be easily broken or formed
- once reaction is complete the products are no longer the right shape to stay in the active site and the complex breaks up.
Induced Fit Hypothesis
- active sites are rigid but flexible
- bonds form between the active sites and substrate which changes the shape of the active site to fit around the substrate to form the active complex.
Enzymes digest and synthesise
- enzymes build substrates in anabolic reactions (which are reactions that synthesise new molecules in a cell)
- enzymes break down/digest substrates in catabolic reactions (which are reactions which break down substrates within a cell)
- combination between anabolic and catabolic reactions makes up our metabolism.
- most metabolic reactions occur as a chain of reactions known as ‘metabolic pathways’
What are intracellular enzymes?
Enzymes that work inside cells
What are extracellular enzymes?
Enzymes that work outside cells. They are secreted by cells and have an effect beyond the boundaries of cell membrane.
Timeline of Enzyme Activity
1835: breakdown of starch for sugar by malt
1877: name enzyme coined to describe chemicals in yeast that ferment sugars
1897: Eduard Buchner extracted enzyme from yeast and showed it could work outside cells
1905: Oho Rohm extracted pancreatic protease to supply enzymes for tanning
1926: James B Sumner produced the first pure crystalline enzyme (urease) and showed enzymes were proteins
1930-1936: Protein nature of enzymes finally established when digestive enzymes crystallised were proteins
1946: Sumner finally awarded a noble prize
What is activation energy?
The minimum amount of energy required to start the reaction (Ea)
How do enzymes reduce activation energy?
Living cells could not survive the temperature needed to make cellular reactions happen fast enough - so enzymes are catalysts that lower the activation energy
Factors that affect enzyme activity
1) Temperature
2) pH
3) Substrate Concentration
4) Enzyme Concentration
(detail in physical cards)
CORE PRACTICAL 1: Investigate factor affecting the initial rate of an enzyme-controlled reaction
IV = the factor (eg. enzyme concentration)
DV = Absorbance (absorbance units)
CV = Total volume of solution in cuvette, total time measuring absorbance (2 minutes) , interval time (10 seconds)
CT = 1 cuvette filled with 4 cm3 of distilled water as the reference cuvette
Another cuvette filled with 2 cm3 of milk and 2 cm3 of distilled water as the testing cuvette
METHOD:
- Dilute the 1% enzyme stock solution with distilled water to produce additional test solutions of 1%, 0.8%, 0.6%, 0.4%, 0.2%. Aim to produce 10cm3 of each concentration.
- Place 2 cm3 of enzyme solution and 2 cm3 of distilled water into a cuvette. This will be used as a reference cuvette for every solution to set the colorimeter absorbance to zero, so we will need five of these reference solutions.
- Then measure 2cm3 of the substrate suspension into a different cuvette and 2cm3 of one of the enzyme concentration solutions using a syringe.
- Take the cuvette with the 2 cm3 of substrate suspension and the syringe with the 2 cm3 of enzyme to the colorimeter so that an accurate initial rate of reaction can be recorded
- Working quickly, mix and place the enzyme into the cuvette containing the milk suspension place that cuvette into the colorimeter and start the stop-clock
- Measure the absorbance immediately and then at 10-second intervals for 2 minutes
- Repeat this for every enzyme concentration solution using a separate reference cuvette for each concentration
- draw graph with absorbance against enzyme concentration
- draw tangent at t = 0
- calculate gradient to find initial rate of reaction
- draw another graph showing
Why is it important to measure the initial rate of reaction?
As that is the point where the rate of different concentration can be comparable with each other, as it is the point where the number of substrates is equal. This is because as the reaction goes on the amount of substrates differs as they are broken down at different rates. By measuring the initial rate of reaction substrate concentration is not becoming a limiting factor as there’s less substrate molecules available to collide with the enzymes.
What are enzyme inhibitors?
Slows down or stops an enzyme from carrying out its function
What are the two main types of enzyme inhibitors?
1) Reversible: a molecule that binds reversibly to the enzyme as they form weak bonds and slows or inhibits the reaction rate
2) Irreversible: a molecule that binds irreversibly with the enzyme and typically form strong bonds with the enzyme. Slows or inhibits the reaction rate permanently.
What are the three things that affect an enzyme?
1) Competitive Inhibitors
2) Non-competitive Inhibitors
3) End-product Inhibition
Competitive enzyme inhibitors
(can be reversible or irreversible)
1) Competitive inhibitors
(compete with the substrate for the active site)
- the inhibitor enters the enzyme environment
- the shape of the enzyme resembles the shape of the substrate
- Due to kinetic energy all of the molecules are moving around
- Sometimes an inhibitor arrives at an active site before a substrate
- The inhibitor binds with the enzyme’s active site preventing a substrate from binding
- Other enzymes active sites are available so a reaction can still occur but just at a slower rate
- If the amount of inhibitor were to increase this would slow the rate further
Non-competitive enzyme (can be reversible or irreversible)
2) Non-competitive (do not bind to the active site but change the shape of the active site)
- The inhibitor enters the enzyme’s environment
- The shape of the inhibitor is different to the shape of the substrate
- The shape of the inhibitor does, however, bind with a different part of the enzyme
- This part is called the ‘allosteric site’
The inhibitor binds to this site, interfering with the bonding holding the tertiary + quaternary and active sites. - The substrate can no longer fit into the active site
- If the inhibitor is reversible, it can dissociate with the enzyme, allowing it to regain its tertiary structure.
Example of competitive inhibitor
malonate —> inhibits succinate
Example of non-competitive inhibitor
Copper sulfate —> inhibits catalase
What is end-product inhibition?`
- it’s a way metabolic pathways can be controlled
- when an end-product in metabolic pathway binds to an enzyme at the start of the pathway
- this process stops the metabolic pathway so prevents further synthesis of the end-product until the end-product concentration decreases
- higher the conc of end-product = quicker the metabolic pathway stops
POISONS
1) snake venom (fasciculin)
- inhibits acetylcholinesterase which is an enzyme used to degrade a neurotransmitter ‘acetylcholine’
- the impulses continue and the muscles go into prolonged spasms causing death as breathing and swallowing becomes impossible
2) Cyanide poisoning (potassium cyanide)
- inhibits a vital respiratory enzyme called ‘cytochrome oxidase’ found inside mitochondria
- any reaction that requires ATP are no longer supplied. The body then has no energy supply causing total cell failure
MEDICINAL INHIBITORS
1) HIV Protease Inhibitors (Protease inhibitors)
- competitively inhibits HIV virus protease enzymes. The inhibitor binds specifically to enzymes which prevent viral RNA pieces from binding so they cannot be implanted into the host cells DNA = no replication
- A host cell can be infected by HIV but cannot be ‘hijacked’ into making more HIV copies as a result of DNA implantation by the virus
2) Suspected antifreeze poisoning treatment (ethanol)
- Ethylene glycol is found in antifreeze, if ingested can be broken down by alcohol dehydrogenase forming an extremely toxic oxalic acid = death
- produces less oxalic acid allowing harmless ethylene glycol to be exerted
