3.0 Enzymes Flashcards
1
Q
What are enzymes?
A
- Biological (function in living systems) Catalysts (speed up the rate of chemical reactions without being used up)
- Globular Proteins : enzymes are proteins with tertiary structure, made of amino acid chains
- every metabolic reaction within living organisms is catalysed by enzymes
- Intracellular : enzymes produced and function inside cell
- Extracellular : enzymes secreted by cells and function outside of cells (e.g. digestive enzymes in gut)
2
Q
Mode of Action of Enzymes (active site)
A
- Active Site : it has a specific shape complementary to its’ substrate, where substrate binds forming enzyme-substrate complex , shape is determined by tertiary structure of protein chain/s (order of amino acid changes, 3D shape changes)
- substrate must collide with enzyme in the right orientation and at the right speed
3
Q
Catabolic vs Anabolic
A
- Catabolic : the breakdown of complex molecules into simpler products
- examples : cellular respiration and hydrolysis
- Anabolic : the building of more complex molecules from simpler ones
- examples: protein synthesis and photosynthesis
4
Q
Mode of Action of Enzymes (Activation energy)
A
- Lowering of Activation Energy : amount of energy needed by the substrate to become just unstable enough for reaction is lowered, bc enzymes influence the stability of bonds in reactants
- they provide an alternative energy pathway
5
Q
Mode of Action of Enzymes (2 hypothesis)
A
- Lock-and-Key theory: globular proteins, 3D shape determined by tertiary structure, therefore highly specific, subsrate fits exactly into active site
- Induced-Fit theory: active site of enzyme can change shape slightly as substrate enters known as conformational change , ensures ideal binding arrangement which maximises ability of enzyme catalysation
6
Q
Methods of Measuring Enzyme Activity
A
- Rate of formation of a product using catalase : hydrogen peroxide (common but toxic by-product of metabolism) must be broken down quickly, catalase breaks it down into water and oxygen, therefore volume of O2 generated can be measured in a set time.
- Rate of disappearance of a substrate using amylase : amylase hyrdolyses starch into maltose and glucose, (best at pH 7 and 37 degrees), combine amylase and starch then test for starch at regular time intervals using iodine in potassium iodide solution (from brown to blue-black in presence of starch) this can be repeated in a variety of conditions (indepedent variables) which is used to compare reaction rates.
7
Q
Use of a colorimeter in enzyme reactions
A
- colorimeter is able to measure light absorbance or light transmission (how much light passes through)
- used in enzyme reactions involving colour change
- As colour breaks down light absorbance decreases and light transmission increases, this is used to mesure the rate of reaction
- range of conc. of stock solution sample are used; serial dilution
- After calibration (adjusting instrument by using a standard substance), % of absorbance and transmission are measured
- values are plotted on a calibration graph
serial dilution
8
Q
The affect of Temperature on Enzyme performance
A
- different enzymes have different optimum temperatures : temp. at which they catalyse a reaction at the maximum rate
-
LOWERING TEMP : prevent or slow down reaction rate
1. molecules move more slowly / speed of enzyme and substrates
2. lower frequency of succesful collision
3. lower rate of enzyme-substrate complex formation
4. substrate and enzyme collide with less energy : less likely for bonds to be formed or broken, stops reaction -
INCREASING TEMP :
1. molecules move more quickly / speed of enzyme and substrates (INCREASE IN KINETIC ENERGY)
2. higher frequency of successful collisions
3. higher rate of enzyme-subsrate complex formation
4. enzyme and substrate collide with more energy (more likely for bonds to be formed or broken) -
BEYOND OP TEMP : denaturation occurs
1. Bonds (hydrogen & covalent) break, changing the tertiary structure of the protein, change in shape of active site (js before denaturation)
2. prevents the complementary subsrate from binding at all (denaturation)
9
Q
The affect of pH on Enzyme Performance
A
- all enzymes have an optimum pH
- enzymes denature at extreme pH values
- Hydrogen and Ionic bonds hold the tertiary structure of protein enzymes
- Excess of H+(acidic) or OH-(alkaline) cause these bonds to break
- Change of active site, decreases rate of enzyme-substrate formation
- when theses complexes cannot be formed at all denaturation has occured
- pH does not affect collision rate, only the ability for substrates to bind to active site; reducing number of succesful collisions
example: pepsin in the stomach functions best at a pH of 2, as HCl is present in the gastric juices
10
Q
Investigating effect of pH on enzymes using buffer solution
A
- Using buffer solutions to measure the rate of reaction at different pH values
- buffer solutions have specific pH value
- they can** maintain this specific pH** even in a reaction
- measured value of buffer solution is added to reaction mixture
11
Q
The affect of enzyme concentration on enzyme performance
A
- affects the rate of reaction
- **higher conc of enzymes, more available active sites, **greater frequency of enzyme-subsrate complex formation
- as long as substrate is available, rate of reaction increases respectively with conc of enzymes
- until rate of reaction remains constant, conc of enzyme is no longer the limiting factor
12
Q
The affect of Substrate concentration on enzyme performance
A
- affects rate of reaction proportionally
- increase in substrate, increase in enzyme-substrate complex formation
- if availability of active site is fixed, increasing substrate conc. will not affect the rate of reaction, instead all active sites are occupied, enzyme-substrate complexes can no longer be formed
13
Q
Types of enzyme Inhibitors
A
- COMPETITIVE INHIBITORS:
- they have similar shapes to that of the specific substrate
- they compete with the substrate for the active site
2.NON-COMPETITIVE INHIBITORS:
- they bind to an alternative site in the enzyme
- this alters the shape of the active site, preventing the substrate from binding
Both types act to slow down or stop enzyme activity. Increasing inhibitor conc reduces rate of reaction.
14
Q
Mode of action of Competitive inhibitors
A
- to counteract the increase of a competitive inhibitor, increasing conc. of substrate can restore rate of reaction
- more substrate molecules increases the frequency of succesful collision, increasing enzyme-substrate formation
15
Q
Mode of action of non-competitive inhibitors
A
- Increasing substrate conc cannot increase the rate of reaction once more
- as the shape of the active site has remain altered, substrates can no longer bind to it
16
Q
reversible inhibitors in metabolic pathways
A
- can act as regulators in metabolic pathways, such reactions must be tightly controlled and balanced so no enzyme goes rouge and produces an excess of a product
- NON-COMPETITIVE REVERSIBLE INHIBITORS: Metabolic reactions can be controlled by the end product of an enzyme catalysed reaction
1. The end product of the reaction acts as the non-competitive inhibitor, when produced it binds to an alternative site in the enzyme
2. reaction has slowed down by itself as the end-product is being produced
3. end-product can detach from the enzyme & can be used elsewhere allowing active site to return to its original shape
4. therefore as product levels fall, enzyme begins catalysing again
5. continious loop, known as End-Product Inhibition
18
Q
Km
A
- the substrate conc at which the enzyme works at half its max rate
- at this point half of the active sites of the enzymes are occupied
- the higher the affinity of the enzyme for substrate, the lower the substrate conc needed for this to occur
- inverse relationship between Km and the affinity of an enzyme for its substrate
- therefore high Km = low affinity and low Km = high affinity
