Enzyme action Flashcards
What are enzymes
Biological catalysts
3 conditions needed for enzyme catalysts
Substrate and enzyme must collide successfully
Energy of products must be less than substrates
Activation energy must be met
Active site
Region which is similar to a substrates shape so the enzyme and substrate can bind
What is formed when an enzyme and substrate bind together
Enzyme substrate complex
Induced fit model of enzyme action
1) Substrate and enzyme collide in a certain way so that the substrate binds to the enzymes active site
2) Active site changes shape to fit with substrate
3) When bind, enzyme substrate complex is formed
4) Bond between molecules breaks
5) Product molecules released from active site
Whole process of how all products are made
1) Lots of substrate, no product
2) Lots of empty active sites, high chance for substrates to collide with active sites
3) All active sites filled and substrate broken down into products
4) Substrate decreases as its broken down, products increase
5) Fewer substrate molecules available so more difficult to collide
6) Rate formation slows, fewer substrate molecules
7) Graph flatterns as substrate has been used up
Effect of temperature
Temperature rises, thermal energy transferred to kinetic energy
Molecules move faster -> more successful collisions, producing more enzyme-substrate complexes
Denatured at around 60C
Effect pH and why it denatures
Denatures = Charges on the amino acids in active site changes, affects the formation of enzyme-substrate complex
This changes shape of the active site
Bonds can also break, changing active site
Effect of substrate concentration
As substrate concentration increases there are more chances of successful collisions, so more enzyme-substrate complexes form
The rate reaches a maximum as all the active sites are occupied (graph platos)
Effect of enzyme concentration
Increases successful collisions, increases rate of enzyme substrate complex production
All substrate is turned into product at the same rate
What are competitive inhibitors
Bind to active site
What are non-competitive inhibitors
Bind to enzyme in position other than active site
Competitive inhibitors and how they act
Compete with substrate for active site
Difference in concentration of substrate and inhibitor, determines effect on enzyme activity
Substrate concentration increases so effect of inhibitor decreases
Longer it will take for product to be made if greater concentration of inhibitor
Non-competitive inhibitors and how they act
Attach at binding site which isn’t active site
Inhibitor alters shape of enzyme where it binds, so active site changes
Substrate can no longer fit
Enzyme cannot function
Inhibitor and substrate are not competing for same site
Concentration increases in substrate, does not decrease effect of inhibitor
What do enzymes change in reactions
Lower the activation energy and catalyse the reaction
Absolute specificity
Enzyme catalyses ONE reaction
Group specificity
Enzyme acts on molecules having specific functional groups like phosphate, amino, methyl groups
Linkage specificity
Enzyme acts on a specific type of chemical bond regardless the remaining molecular structure
Stereochemical specificity
Enzyme acts on a certain optical or steric isomer
How is specificity attained
When the substrate interacts weakly with the enzyme’s active site to form a bond
Producing covalent bonds that the enzyme can only catalyse
What do enzymes catalyse
Wide range of intercellular and extracellular reactions
Determining structures and functions of a whole organism
What two things can you measure enzyme activity (practical)
Measure rate of formation of a product using catalase
Measuring rate of diappearance of substrate using amylase
What is measured for the investigation of amylase activity using iodine
Take samples from reaction mixture at each time interval and adding each sample to iodine in potassium iodide solution
Starch = blue-black
No starch = yellow-brown
Why use amylase-starch solution
Amylase = digestive enzyme that hydrolyses starch into maltose and glucose
Method
1) Place single drops of iodine solution in rows on tile
2) Label test tube with pH to be tested
3) Use syringe to place 2cm^3 of amylase in test tube
4) Add 1cm^3 of buffer to test tube using syringe
5) Add 2cm^3 using different test tube of starch solution to amylase and buffer solution, start stopwatch whilst mixing using pipette
6) At 10 secs, use pipette and place on e drop of mixture on first drop of iodine (should turn blue black)
7) Wait another 10 secs and place another drop of mixture on second drop of iodine
8) Repeat every 10 secs
9) Repeat at different pH values
Less time iodine solution takes to remain orange-brown, quicker starch has been digested , better enzyme works at that pH
What proteins are enzymes
Globular proteins
What pathway is controlled by enzymes
Metabolic pathway
Are enzymes active inside or outside the cell
Intraceullar or extracellular
INtracellular = produced and function inside cell
Extracellular = secreted by cells and catalyse reactions outside cells
What is the shape of the active site determines by
1) Proteins formed from chains of amino acids held by peptide bonds
2) Order of amino acids determines shape of enzyme
Two type of enzyme reactions
Catabolic and anabolic
WHat are catabolic reactions
Breakdown of complex molecule substrate into simpler products
When a single substrate is broken apart into two or more products
E.g. cellular respiration and hydrolysis
What are anabolic reactions
Building more complex molecules from simpler ones
Two substrates bond to form one single product
What is the activation energy
The amount of energy needed by the substrate to become unstable enough for a reaction to occur and for products to be formed
How do enzymes speed up chemical reactions
Influence the stability of bonds in reactants
The destabilisation of bonds in the substrate make them more reactive
How can you measure the progress fo enzyme catalysed reactions
Measuring rate of formation of a product = catalase
Disappearance of a substrate = amylase
How is catalase used to investigate the rate of product formation
Hydrogen peroxide is a toxic by product f metabolism
It must be broken down quickly
Catalase is found in cells so you can break down hydrogen peroxide into water and oxygen
Hydrogen peroxide and catalase are combined and the volume of the oxygen generated is measured
How do you investigate amylase using iodine
Amylase hydrolyses starch into maltose and glucose
Amylase and starch combine and this reaction mixture is then tested for starch at regular time intervals
Take samples from the reaction mixture at time intervals by adding iodine in potassium iodide solution
Starch forms blue black, no starch yelliw brown
Investigating the effect of starch concentration on amylase using colourimetry
Colourimeter measures light absorbance
As colour breaks fown light absorbance decreases
1) Calibration calorimeter (use weak iodine solution)
2) Prepare starch solution of known concentration from which a range of concentrations are made using serial dilutions
3) Use with different concentrations of starch
4) Calibration graph plotted
What do lower temps do
Lower frewuency of successful collisions between substrate and active site
Molecules move slower
Less frequent enzyme substrate complex formatos
Substrate and enzyme collide with less energy so bond formation is less likely
What happens when temperatures rise too high
Bonds holding enzyme mlecule in precise shape break
Tertiary structure of protein changes
Active site permanently damages preventing substrate binding
Denaturation occurs is substrate can no longer bind
What happens at extreme pH changes
Is solutions with excess H+ and OH- ions can cause hydrogen and ionic bonds to break
Alters shape of active site = less ESC
ESC can no longer form at all
Complete denaturation occurs
Buffer solutions affects from different pH
Buffer solutions each have a specific pH
Buffers maintain pH
Measure volume o fbuffer added to reaction
Same volume should be added for each pH that is being investigated
How to calculate pH
PH = log [H+]
How do enzyme concentrations affect rate
Higher conc = greater number of active sites available and greater likelihood of ESC
As long as there is a sufficient substrate available, rate of reaction increases linearly
Otherwise will not increase and is a limiting factor
How does substrate conc affect rate of reaction
Number of sub molecules increases likelihod f ESC increases
ENcyme conc fixed but substrate increased past a certain pint all available active sites eventually saturated so rate of reaction cant increase
WHen active sites of enzymes are full subs have nowhre to bind
Linear increase then plateaus
What are reversible inhibitrs
Regulators in metabolic pathways
Metabolic reactions are tightly controlled and balanced so that no single enzyme can cntinuously and uncontrollably generate more and more of a particular product
How are metabolic reactions controlled
Using the end prduct f a particuar sequence of metabolic reactios as a non competitive reversible inhibitor
Process of end-product inhibition
1) Enzyme converts substrate to product
2) End product of reaction chain binds to alternative site on enzyme, changing active site shape preventing formation of further enzyme substrate complexes
3) end product detaches from the enzyme and used elswhere allowing thr active site to reform
What does an enzyme to do the reaction
1) Lowers AE and provides alternative pathway for substrates to react on
2) Destablises bonds of substrate making it more reactive, decreasing energy needed to break them
2 ways an enzyme might reduce actuation every
Holding 2 substrates close together in the enzyme-substrate complex and making it easier for substates to collide
By stressing bonds within the substrates and making it easier to break then
What occurs in the induced fit hypothesis that DOESN’T occur in the lock and key
1) enzyme structure can move
2) Active site and substrate are not a perfect complementary fit to one another
3) Active site changes shape to fit the substrate
4) This weakens the bonds of the substrate and lowers the activation energy
How can an enzyme denature
High temp
Extreme low or high pH
Cause weak bonds in tertiary structure to break changing the shape of the active site
Key terms
Denature
Active site, complimentary, enzymesub complex
destabilises bonds of substrate making them more reactive
Mention compet or non competitive inhibitor
Similarities and differences between competitive and non competitive inhibition (6 points)
Similarities:
-Inhibitor binds to surface
-Reduce enzymes rate
-As inhibitor conc increases rate of reaction decreases
Differences:
-Compet inh binds to active site, non compet binds to allosteric site
-Active site blocks in competitive, active site distorted in non compet
-increase sub conc will decrease effect of compet inhibition nut will not affect non-compet inhibition
Differences and similarities between substrate and competitive inhibior
Similar:
-Similar shape so both are complementary to enzymes active site
Differences:
-Competitive inhibitor may stil bind to the enzyme whereas a product will always unbind from the enzyme
-No product is formed with a competitive inhibitor
What does specificity mean
One enzyme has a small number or substrates only
Due to exact complementary shape between the substrate and active sites
Why does the reaction between an enzyme and substrate occur at 37 degrees C (for example could be any temp)
Sucrase lowers the activation energy required
By stressing the substrate forming an enzyme substrate complex
