Enzymes Flashcards
How can the Enzymatic reaction rates be calculated?
The rate of the reaction is the amount of product formed in a certain amount of time, which is identical to the amount of reagent that disappear in that time.
v=d[P]/dt=-d[A]/dt
The rate constant k is determined by the free energy difference ΔG, which determines the direction of the reaction, and by the activation energy ΔG++, which determines the rate of the reaction since it is the energy barrier that needs to be overcome to produce products.
What are enzymes? How do they work?
Enzymes are a class of proteins that catalyse all reactions in the human body. To accelerate the rate of the reaction, it lowers the activation energy.
What is the mechanism of catalysis?
- The substrate bind to the binding site, forming the enzyme-substrate complex.
- The enzyme brings the reagents close together, prevent the reagents from diffusing away and ensure that they are in the correct orientation. The complex is now called transition-state complex.
- the product is release and the enzyme is free.
For some enzymes, the interaction between enzyme and substrate is sufficient for lowering the activation energy; other needs other factors that induce the formation of the products:
- Acid-Base catalysis: acceleration of the reaction due to changes in the pH, which induce changes in the substrate. To break the the enzyme can either:
- hydrolyse the peptide bond
- Covalent catalysis: the bond within the reagent is replaced by a bond between enzyme and substrate, and then broken.
Which parts form the Active site of an enzyme?
The active site is the place where the substrate binds to.
It is divided in:
1. Binding site: bind and properly orient the substrate
2. Catalytic site: reduce the activation energy barrier
What are the substrate binding mechanisms?
- Lock-and-Key: the substrate fit into the pre-formed active site of an enzyme
- Induced Fit: the substrate binds to the enzyme and induce a single specific conformation
What are cofactors? How can they be discriminated?
Cofactors are non-protein chemical compounds that are required for an enzyme’s activity.
- Inorganic:
- electron carriers
- Organic/coenzyme:
- redox cofactors
- activation-transfer coenzymes
What is the Michaelis-Menten equation? Why is it used?
The Michaelis-Menten equation is used to calculate the rate of an enzyme, by considering the relation between the maximum rate, the substrate concentration and the equilibrium constant for binding.
v=(vmax*[S])/(Km+[S])
How is enzymatic activity regulated?
Enzymatic activity is regulated by Inhibitors, compounds that reduce the activity of an enzyme:
1. Competitive inhibitor: competes with the substrate by binding in the active site of the enzyme.
vmax is not affected but Km is.
2. Uncompetitive inhibitors: only binds to the enzyme-substrate complex.
both vmax and Km are affected.
3. Non-competitive inhibitors: bind equally well to the enzyme and to the enzyme-substrate complex.
vmax is reduced, Km is not affected.
4. Mixed inhibitors: bind to enzyme and enzyme-substrate complex with different affinity.
vmax is reduced, Km is increased.
What are irreversible mechanisms?
- Irreversible inhibition: covalent binding of a functional group to the active site of an enzyme.
- Irreversible activation: zymogens
What are reversible modifications?
Reversible modifications are modifications that involve covalent bonds:
1. Phosphorylation: transfer of a phosphate from ATP, causing either the activation or deactivation of the enzyme.
2. Histone acetylation: remotion of positive charges to release DNA and enable gene transcription.
3. Histone Methylation: methyl group bind to histones, increasing the DNA binding and silencing gene transcription.
4. Ubiquitination:
- Mono-ubiquitination: affect proteins localisation and interaction
partners.
- Poly-ubiquitination:
- via Lys48: target a protein for degradation
- cia Lys 63: alter cellular localisation and interaction
What is meant by cooperativity?
Cooperativity is the binding of substrate to one subunit that change the conformation and affinity of the other subunits for that substrate.
These substrate affect the equilibrium between tension and relax state.
What is allosteric regulation?
- Allosteric inhibition: occur when a molecule increases the activity of a protein by binding to it at a different place than the substrate, stabilising the T-state.
- Allosteric activation: occur when a molecule decreases the activity of a protein by binding to it at a different place than the substrate, stabilising the R-state.
How does oxygen binding occur?
Oxygen binds to the Heme group of either hemoglobin, responsible for the transport of oxygen via blood, or myoglobin, responsible for the storage of oxygen.
As oxygen binds to the heme group, it causes a conformational change in the hemoglobin/myoglobin:
1. Tense-state: oxygen-free conformation
2. Relaxed-state: oxygen-bound conformation
This is an example of cooperativity, since, as oxygen binds to one subunit of the -globin, the oxygen affinity of the other three is increased.
