05-10-21 - Introduction to Enzymes Flashcards
Enzymes that are biological catalysts
• How are they chemically altered in reactions?
• What is their function?
• How do they do this
• What conditions do they work in?
• How do they work?
What are they specific to?
• Properties- Enzymes in any reaction are not chemically altered in a reaction.
- Their function is to increase the rate of reaction by providing a pathway of lower activation energy to get reactants to products
- They do this by stabilising the transition state.
- They operate under physiological conditions (around 37°, neutral, low substrate concentrations, aqueous environment
- Biological catalysts work by forming complexes with their substrate (binding), thus providing a unique microenvironment for the reaction to process
- Enzymes contain an active site where the substrate binds to.
- Enzymes have very high specificity for both the reaction catalysed and substrate used
- The activities of some enzymes are regulated in the body.
What is rate enhancement of a catalyst?
How is it calculated?
- Rate enhancement of a catalyst is the factor by which the catalyst increases the rate of reaction
- Rate enhancement = catalysed rate/uncatalyzed rate.
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What is an example of a biological catalyst?
What does it do?
How does it work?
- Lysozyme catalyses the cutting of polysaccharide chains
- It binds to polysaccharide chains, catalyses the cleavage of the specific covalent bonds, and released the cleaves products
- It remains unchanged at the end of the reaction.
What are the 4 reasons why the accumulation of products is not linear on a graph?
What is the initial reaction velocity (Vo)?
What is it used for?
- Product accumulation is not linear because:
- Substrate concentration falls
- Products may inhibit the enzyme
- Enzyme might denature
- Reverse reactions may become more favourable
- The initial reaction velocity (Vo) is the initial rate of reaction before any of the factors that slow the rate of reaction become significant.
- Initial rate is what we measure when trying to calculate enzyme activity, and is the only valid parameter.
What is the activity of an enzyme dependent on?
How is enzyme activity (Vo) measured?
Why do we do it this way?
- The activity of an enzyme depends on how rapidly it can process a substrate.
- Enzyme activity (Vo) is measured by increasing the substrate concentration and measuring the accumulation of products over time.
- This is so the reaction rate is not limited by decreasing substrate concentration.
How is rate of reaction linked with substrate concentration?
What is V max of an enzyme?
How can it be calculated?
- The rate of reaction increases as the substrate concentration increases, until a maximum is reached (Vmax)
- Vmax is the maximum reaction velocity of an enzyme
- The Vmax of an enzyme is difficult to reach, so it can be calculated by plotting a Lineweaver-Burke plot (double reciprocal of the data)
- This produces a straight line, where the y intercept on the graph is equal to 1/Vmax (reciprocal of y-intercept = Vmax)
What is Km? How is the Michaelis-Menten constant (Km) calculated?
What is it used for?
What is the equation for initial reaction velocity?
- Km is the substrate concentration required for half maximum reaction velocity (Vmax)
- The Michaelis-Menten constant is used in conjunction with Vmax to measure the activity of an enzyme using the Michaelis-Menten equation.
What is the biological significance of Km in terms of hexokinase and glucokinase?
- Hexokinase and Glucokinase both catalyse the phosphorylation of glucose, but they have different Km values
- Hexokinase has lower Km and phosphorylates glucose at low concentrations of glucose
- It is found in all tissues and is required for energy production in cells
- A low Km ensures the utilisation of glucose, even at very low concentrations
- Glucokinase has a higher Km.
- It is found in the liver
- Glucokinase will only phosphorylate glucose when blood glucose concentrations are high, this allows for the production of glycogen, which can be stored
- A high Km ensures glucose is not removed from the blood for storage at low concentrations.
How is Km determined?
• Km is determined by using a Lineweaver-Burke plot and dividing the Vmax by 2. The X intercept is also equivalent to -1/Km. If we take the reciprocal of the X intercept, we will have the Km value.
What are enzyme inhibitors?
What are the 2 types of enzymes inhibitors?
- Enzyme inhibitors are chemicals that interfere with enzyme reactions.
- There are irreversible and reversible inhibitors
- Irreversible inhibitors include inactivators
- Reversible inhibitors include competitive and allosteric (Non-competitive)
- Competitive inhibitors compete with the enzyme’s substrate for access to the active site
- Allosteric enzymes bind to another part of the enzyme which is not the active site.
How does irreversible inhibitors work?
What is an example of irreversible inhibition?
- Irreversible inhibitors react with the enzyme and form a covalent adduct with the protein
- This inactivation is irreversible
- DIPF is an organophosphate pesticide, which inhibits AChE
- AChE is an enzyme that degrades neurotransmitter Ach into choline and acetic acid
- Organophosphates inactivate AChE by phosphorylated the serine hydroxyl group located at the active site of AChE
- This inactivation causes ACh accumulation throughout the nervous system, resulting in overstimulation of receptors, which is fatal to pests.
What is aspirin?
How does aspirin work as an irreversible inhibitor?
- Aspirin is an antipyretic (reduces fever), anti-inflammatory, and analgesic (painkiller)
- It works by irreversible inhibiting COX-1
- COX-1 catalyses the conversion of AA (arachidonic acid) to Prostaglandin H2 (precursor for synthesis of inflammatory mediators)
- Aspirin reacts with a serine residue close to the active site of COX-1, preventing the substrate AA from binding
How does competitive (reversible) inhibition work?
How are competitive inhibitors structured?
How is the action of an enzyme affected by competitive inhibitors?
- A competitive drug can compete with the substrate for the active site of the enzyme.
- The substance has a similar structure to that of the normal substrate.
- The drug will occupy the active site and then leave it unchanged.
- The action of the enzyme is slowed down by the presence of a competitive inhibitor.
- A competitive inhibitor is like a key that gets though the keyhole but cannot unlock the door.
How do competitive inhibitors affect Vmax and Km of an enzyme? How do competitive inhibitors affect graphs and Lineweaver-burke plots?
- A higher concentration of substrate is needed to reach Vmax, as more substrate is needed to outcompete the competitive inhibitor
- This leads to Vmax being unchanged, but an increase in Km, as Km is the substrate concentration needed to reach half of Vmax
- On graphs, the curve is extended to the right, as a higher substrate concentration is needed to reach Vmax
- On line-weaver burke plots, the y-intercept is the same, as the Vmax is the same, but the gradient is steeper, as a higher substrate concentration is needed to reach Vmax.
When can allosteric inhibitors bind to enzymes?
What do allosteric inhibitors to do enzyme?
What won’t help reactivate the enzyme?
What happens to Vmax and Km values due to allosteric inhibitors?
- Allosteric inhibitors can bind to the enzyme at the same time as the substrate, because they never bind to the active site.
- Allosteric enzymes bind to the enzyme, and alter its active site, so the substrate can no longer fit inside it.
- This makes the enzyme inactive
- Increasing substrate concentration cannot drive away the allosteric inhibitor from the enzyme, so this does not help increase the rate of reaction (in contrast to competitive inhibition)
- Vmax decreases
- Km often (but now always) increases
What are the 2 types of allosteric inhibition?
What effects do they have on Vm and Km?
non-competitive inhibition:
- Inhibitor binds independently of substrate and reduces the efficacy of the enzyme
- Vmax decreases due to reduced efficiency of the reaction
- Km (and substrate affinity) is unchanged
mixed inhibition:
- Inhibitor binds allosterically but not independently of substrate
- Inhibition can be reduced but not overcome by increasing [S]
- Vmax decreases due to inhibition
Km increases as substrate affinity is reduced
What is an example of allosteric inhibition?
How is this also an example of feedback inhibition?
- The enzyme PFK catalyses the transfer of phosphate to fructose-6-phosphate
- This is an important step in glycolysis (process that produces positive net ATP)
- PFK binds ATP at two sites (an allosteric inhibitory site and the active site)
- In the presence of high ATP levels, the inhibitory site is occupied, and fructose-6-phosphate binding is affected
- This prevents glycolysis from proceeding when ATP is not needed.
- This is known as feedback inhibition, where a product of the pathway acts as an inhibitor to prevent the process from continuing.
How is enzyme kinetics used to treat anti-freeze poisoning?
- The toxin responsible for anti-freeze poisoning is oxalate, which is produced when ethylene glycol from the anti-freeze is broken down by alcohol dehydrogenase
- By administering a near-intoxicating dose of ethanol to the patient, this allows ethanol to act as a competitive inhibitor against ethylene glycol, which prevents oxalate from getting made.
