protein function and enzymes Flashcards
Haemoglobin
-transport oxygen in the blood binds in lungs releases in capillaries
-haemoglobin is tetrameric
-means it has 4 polypeptide chains
-2 alpha chains 141 amino acids
-2 beta chains 146 amino
-each chain folds to form 8 alpha helices
myoglobin
-stores oxygen in muscles – skeletal and cardiac
- monomeric
-is a single polypeptide chain of 153 amino acids folded to form 8 alpha helices
-haem prosthetic group – protoporphyrin IX, with central Fe2+ atom
-higher affinity for oxygen
haem prosthetic group
-oxygen binds to haem prosthetic group
binding in haemoglobin
When oxygen binds to one of the subunits, it triggers a change in the shape of that subunit.
This conformational change makes it easier for oxygen to bind to the other subunits of hemoglobin.
This effect is called cooperative binding
Tense (T) state: In the deoxygenated form, hemoglobin is in a “tense” state, where the subunits are less likely to bind oxygen.
Relaxed (R) state: When oxygen binds, hemoglobin undergoes a conformational change from the T state to the relaxed (R) state, which has a higher affinity for oxygen.
Hemoglobin is considered an allosteric protein because its function (oxygen binding) is regulated by the binding of oxygen to different sites on the protein.
details of conformational change
-fe2+ is the central atom in the heam group is slightly out of place but when the oxygen binds it moves back into the plane
which also pulls the His F8 ligand and the F helix, causing a small but significant structural change (0.029 nm).
This change in one subunit facilitates the binding of oxygen to the other subunits, demonstrating allosteric regulation and cooperative binding, where the binding of oxygen to one subunit makes it easier for other subunits to bind oxygen.
pH and Bohr effect
-Binding of protons by haemoglobin lowers its affinity for oxygen contributing to a phenomenon known as theBohr effect
-CO₂ produced by metabolism enters red blood cells and is converted to carbonic acid by carbonic anhydrase.
-The dissociation of carbonic acid releases protons (H⁺), which lower the pH of the blood.
-These protons bind to hemoglobin, causing conformational changes that reduce hemoglobin’s ability to hold oxygen, thereby promoting oxygen release into tissues where it’s needed. This process is known as the Bohr effect.
enzymes
-enzymes lower the energy of the transition state of a chemical reaction
-highly specific and only work under specific conditions
-Enzymes use functional groups at the active site provided by amino acids and might also include coenzymes or metal ions
-
enzymes and thermodynamics
To start a biochemical reaction, molecules must overcome an energy barrier to reach an unstable transition state this is where there is the most free energy its part way between substrate and product . The energy required to reach this state is called the Gibbs free energy of activation (ΔG‡). (the difference in free energy of substrate and transition state.
Enzymes help by stabilizing the transition state, lowering the activation energy, and speeding up the reaction. This is achieved through mechanisms like entropy reduction, proper orientation, distortion of substrates, and solvation of the transition state.
, enzymes do NOT change the difference in free energy between substrate and product
maximum velocity (Vmax)
when all enzyme molecules are occupied by substrate (in the E-S complex), and increasing substrate concentration further has little effect on the reaction rate
hyperbolic curve
A plot of the initial velocity (V₀) versus the substrate concentration ([S]) produces a hyperbolic curve. This reflects the enzyme’s behavior at low and high substrate concentrations.
At low [S], V₀ increases proportionally with [S].
At high [S], V₀ approaches Vmax and levels off.
Michaelis-Menten - Lineweaver-Burk plots
The Michaelis constant (Km) is the substrate concentration at which the reaction rate is half of Vmax, and it reflects the enzyme’s affinity for its substrate (lower Km = higher affinity).
To analyze enzyme kinetics more easily, the Lineweaver-Burk plot is used to transform the data into a straight line, making it easier to determine both Vmax and Km.
two ways of enzyme bonding
-lock and key model
-induced fit model (requires conformation change)
ways enzymes lower the transition state
-Acid –Base (proton donation/abstraction)
-Temporary covalent bond formation
-Redox effects
-Electrostatic effects
-Orientation/proximity effects and straining effects.
cofactors
Cofactors are non-protein molecules that are required for a reaction to take place in addition to the enzyme and the substrate
Cofactors can be organic (e.g. ATP, NADH) or inorganic (e.g. Mg2+, Zn2+)
-
cofactors split into two sub categories
Cofactors that bind loosely and are chemically altered by the enzyme are also called coenzymes. They are distinguished from substrates by being recycled for participating in the same reaction(s).
Cofactors that bind tightly are considered part of the enzyme. In this case the enzyme without the cofactor is called the apoenzyme and with the cofactor it is called the holoenzyme.
competitive inhibition ; regulation
-By competitive inhibition: a molecule competes with the substrate for the substrate binding site.
Many drugs work this way. e.g., statin mediated inhibition of cholesterol synthesis.
Can be reversible or irreversible
Also have non-competitive inhibition (see pharmacology lectures)
-
covalent modification ; regulation
Covalent modification is a form of enzyme regulation where the activity of the enzyme is controlled by the addition or removal of specific chemical groups to/from its amino acid residues. This can result in either an increase or decrease in enzyme activity, depending on the modification.
Common types of covalent modifications include:
Phosphorylation (addition of a phosphate group, usually to serine, threonine, or tyrosine residues),
Methylation (addition of a methyl group, typically to lysine or arginine residues),
Acetylation (addition of an acetyl group, often to lysine residues).
example of covalent modification
e.g. aspirin acetylates cyclooxygenase and inactivates it
allosteric regulation
Allosteric regulation occurs when a molecule binds to an enzyme at a site other than the active site, causing a conformational change that affects enzyme activity. This binding can either increase or decrease enzyme efficiency. It is a common form of regulation, where, for example, the product of an enzyme can inhibit its own activity. some cases, a regulatory protein can bind to the enzyme and influence its activity.
This regulatory protein, often called a modulator, can either enhance or inhibit the enzyme’s function, acting as an additional layer of control over enzyme activity.
Why are enzyme mechanisms important for pharmacy and pharmaceutical sciences?
with examples
Enzymes are common drug targets because they affect large numbers of substrate molecules: a small change in enzyme activity leads to a large change in the product concentration
-Anti-retroviral protease inhibitors used to treat HIV/AIDS
-Statins inhibit enzymes involved in cholesterol metabolism
-Non-steroidal anti-inflammatory drugs (NSAIDs) modulate pain and inflammation by inhibiting cyclooxygenases
-Antibiotics like penicillin block enzymes involved in assembly of bacterial cell wall
