Flashcards in Topic 7 - Sigmoidal Kinetics - Hemoglobin & Myoglobin Deck (19):
Sigmoid (S-shaped) kinetic curves: Slope at very low [S] suggests low affinity for S (i.e., a very high [S] to reach 1/2 Vmax). Indicates that the enzyme will have a higher affinity for substrate at higher [S] concentrations.
Low affinity conformation, may not bind substrate (O2).
DeoxyHb will be T-state.
High affinity conformation, binds substrate (O2).
Binding of substrate increases stability of R-state.
Oxy-Hb will be R-state.
Koshland - Sequential Model
This model for allosteric regulation of enzymes suggests that the subunits of multimeric proteins have 2 conformational states.
Binding of the ligand causes conformational change. Although the subunits go through conformational changes independently (as opposed to in the MWC model) the switch of one subunit makes the other subunits more likely to change, by reducing the energy needed for subsequent subunits to undergo the same conformational change. In elaboration, the binding of a ligand to one subunit changes the proteins shape, thereby making it more thermodynamically favourable for the other subunits to switch conformation to the high affinity state (R-state).
Monod-Wyman-Changeux - Concerted Model (Symmetry Model)
This model for allosteric form of cooperativity suggests that an oligomeric protein can exist in two conformational states in the absence of the ligand. These states are in equilibrium, and the one that is predominant has a weaker affinity for the ligand (which binds to the protein in a rapid equilibrium fashion).
The homotrophic effect of an allosteric enzyme is seen when the SUBSTRATE acts as an effector and alters the activity of the enzyme (the substrate binds to the active site and induces allosteric-like effects.)
[When enzymes exhibit positive cooperativity of substrate binding to the multiple active sites of these multimeric enzymes.]
The heterotropic effect of an allosteric enzyme is seen when an EFFECTOR modifies the activity of the enzyme.
[Many enzymes also have allosteric binding sites for regulatory molecules (positive and negative effectors) that stimulate or inhibit enzyme activity.]
Describe the process of Hb to lungs and muscle cells.
deoxyHb (Hemoglobin w/o O2) travels to lungs to pick up O2, then travels to muscle cell to give the O2 to Myoglobin, which stores the O2 until needed.
Why do Hb & Mb have different binding curves?
Myoglobin must be able to bind O2 right away, has a steeper binding curve than Hemoglobin (which resembles sigmoidal kinetics). This is because Hb must bind O2 in the lungs (where the pressure of O2 is high) and then drop it off in the muscle cells (where the pressure of O2 is low).
What is the Oxygen binding site for both Mb & Hb?
It is the same type of heme prosthetic group - the protoporphyrin IX coordinated to Fe2+.
How are the structures of Mb & Hb different?
Myoglobin is a monomer while Hemoglobin is a tetramer. Their tertiary structures look identical however.
What is the difference in structure between deoxyHb and Hb? (Before and after binding O2)
DeoxyHb has more space between beta sidechains (in the middle). After binding O2, it rotates 15 degrees and brings the beta sidechains closer together, shifting the sidechains and resulting in a conformation from the T-state (unbound) to the R-state (bound) due to new bond changes.
How does the structure change after Hb binds O2? What must it pick up on the way back and why?
In the lungs: Binding O2 changes the porphyrin ring. It will pull the proximal His, which will pull the F helix and induce a conformational change. All subunits change to R (concerted model) and will now be stabilized by new bonds.
It then carries the O2 to the tissues and releases it. Hb becomes protonated in order to prevent picking up O2 on its way back.
What happens if Carbon Monoxide binds to Hb instead of O2?
If Carbon Monoxide (C=O) binds instead of O2 (O=O), it increases Hb affinity for O2 (of other subunits), causing a tighter R-state. Hb will not drop off O2 to tissues.
Negative Effectors of O2-binding to Hb
1. D-2,3-bis Phosphoglycerate (2,3-BPG)
2. The Bohr Effect: H+ (pH) & CO2
3. CO2 forms carbamates w/ free alpha-amino groups of Hb beta-chains to stabilize deoxy-Hb in tissues and help transport more CO2 to lungs where it is exhaled.
4. Cl- concentration in RBC of venous blood is higher than in arterial blood. Cl- is a negative effector of oxygen binding, binds preferentially to deoxy-Hb.
How does D-2,3-bis Phosphoglycerate (2,3-BPG) act as a negative effector of oxygen binding to Hb?
Hb curve without BPG is steeper (like Mb) than with BPG, so Hb without BPG has a higher binding affinity and therefore more likely to bind O2. Without BPG, Hb can pick up O2 but cannot drop it off in the tissues.
BPG has negatively-charged residues due to its Phosphates.
BPG binds in the center pocket of Hb, O2 binds in the outer.
In normal adult blood, there is a Histidine in Beta-1 and Beta-2. In fetus blood, there is a Serine instead of a Histidine to take away the positively-charged residues that would bind BPG, which stabilizes the T-state. In fetus blood, it is therefore considered a Gamma subunit instead of a Beta subunit. This reduces the affinity for BPG and increases affinity for O2 for fetal blood! Baby Hb is different from the mom's Hb so that O2 can be transferred from the mother to the fetus. Baby Hb has a higher binding affinity for O2 (because it doesn't have BPG bound to it) so it steals the O2 from its mother. When the baby is born, it stops expressing the Gamma chain and starts expressing the Beta chain.
Lets us breathe at higher altitudes!
2. The Bohr Effect: H+ (pH) & CO2
3. CO2 forms carbamates w/ free alpha-amino groups of Hb beta-chains
CO2 forms carbamates w/ free alpha-amino groups of Hb beta-chains to stabilize deoxy-Hb in tissues and help transport more CO2 to lungs where it is exhaled.