bioch lec 4
What determines a protein’s function and 3D structure?
Function is dictated by structure; 3D structure is determined by primary (1°) sequence.
Myoglobin vs hemoglobin—quaternary structure?
Myoglobin (Mb) is a monomer (no 4°). Hemoglobin (Hb) is an oligomer (4°).
Where do Hb and Mb operate?
Hb binds O₂ in lungs and releases in tissues; Mb binds O₂ in muscle cells.
Functions of myoglobin?
Facilitates O₂ diffusion, local O₂ reserve during intense exercise, O₂ storage in aquatic animals.
What is Kd?
Dissociation constant:
K
d
=
[
P
]
[
L
]
[
P
L
]
K
d
=
[PL]
[P][L]
; lower Kd = higher affinity.
Shape of a simple ligand-binding curve?
Hyperbolic (fractional saturation vs [ligand]).
Two ligands, different curves—what differs?
Affinity (Kd); lower Kd reaches saturation at lower [ligand].
Structural highlights of myoglobin?
153 aa, 8 α-helices, heme in hydrophobic pocket between E & F helices.
What is heme?
Planar porphyrin with Fe²⁺ coordinated to 4 ring nitrogens; propionyl groups are polar, rest largely hydrophobic.
What anchors heme in Mb?
Hydrophobic pocket + coordination to His F8 (proximal His); this helps prevent Fe²⁺ oxidation.
Which Fe²⁺ coordinate does proximal His (His F8) occupy?
5th coordination position of Fe²⁺.
Where does O₂ bind on heme?
6th coordination position of Fe²⁺.
Role of distal His (His E7)?
Assists O₂ binding, raises O₂ specificity, lowers CO affinity (improves selectivity).
Do Mb’s E/F helices move on binding?
No; Mb shows constant affinity behavior.
Mb + O₂ equilibrium & Kd meaning?
Mb+O 2⇌MbO 2
; at pO₂ = Kd, Mb is 50% saturated (hyperbolic curve).
Hemoglobin composition?
α₂β₂ heterotetramer; each subunit has heme between E & F helices (like Mb).
Relationship of α, β, and Mb?
Homologous folds (8 α-helices + pocket) with conserved key residues.
Mb vs Hb oxygen-binding mechanism?
Identical core chemistry: O₂ at Fe²⁺(heme); His F8 and His E7 are invariant and critical.
Conservative vs critical substitutions?
Conservative (e.g., Leu↔Ile) ≈ minor effects; critical (e.g., Ser↔Val) can alter function.
What does a hyperbolic curve indicate?
Constant affinity (e.g., Mb).
What does a sigmoidal curve indicate?
Cooperative binding with changing affinity (e.g., Hb).
Mb vs Hb core roles?
mb:O2 handling within tissues. Hb: O2 transport lungs--> tissues
Hb’s two conformations?
T (tense, low affinity) and R (relaxed, high affinity); cooperativity enables efficient delivery.
What changes on oxygenation?
Histidine positions shift at interfaces; central cavity shrinks (R) from larger (T).
Define allostery & effector types.
Effector binding changes affinity at other sites: homo (same ligand), hetero (different ligand); activators increase, inhibitors decrease affinity.
In Hb, what is O₂’s allosteric role?
Homoallosteric activator—binding O₂ promotes more O₂ binding (positive cooperativity).
Sequence of events when first O₂ binds Hb?
Fe²⁺ moves into heme plane → His F8 & helix F shift → subunit interfaces change → raise affinity in other subunits (T→R).
How do heteroallosteric effectors show up on curves?
Activators shift to higher affinity, inhibitors to lower affinity (O₂ curve remains sigmoidal).
Name Hb effectors.
O₂ (homo-activator); 2,3-BPG & H⁺ (hetero-inhibitors for O₂).
Why is 2,3-BPG essential?
Stabilizes T-state, reduces O₂ affinity → enables release in tissues.
Where does 2,3-BPG bind?
Central cavity of deoxy (T) Hb; interacts with positively charged residues (~4 His, 2 Lys, 2 N-termini). The R-state cavity is too small.
How do protons (H⁺) affect Hb?
Metabolism → H⁺ (ATP hydrolysis, CO₂ hydration). Protonation enhances BPG binding, reduces O₂ affinity; alters subunit interfaces → Bohr effect.
Lungs vs active muscle: what favors O₂ binding/release?
lungs: high PO2 and higher pH (7.6) --> R state, O2 binding
tissues: low pO2 and lower pH (7.2) --> T state, O2 release
Net message?
O₂, pH (H⁺), and 2,3-BPG together set the T↔R equilibrium to match physiological needs.
Why do substitutions matter?
Structure absolutely determines function; mutations can be pathogenic (e.g., sickle cell) or adaptive (fetal Hb).
What is the sickle mutation & its effect?
β6 Glu→Val (polar→hydrophobic). In T-state, a hydrophobic patch is exposed; Val fits and promotes polymerization into fibers → sickled cells.
Subunit composition & key change?
HbF = α₂γ₂; γ ≈ β but His143→Ser reduces BPG binding → higher O₂ affinity (helps extract O₂ from maternal blood).
Name the critical His roles in Hb.
His F8 (proximal): heme attachment & protects Fe²⁺ from oxidation.
His E7 (distal): assists O₂ binding, lowers CO affinity.
4 His in central cavity: 2,3-BPG binding.
His at interfaces: contribute to T↔R electrostatics.
Why does Hb show a sigmoidal O₂ binding curve while Mb is hyperbolic?
Hb undergoes T↔R allosteric transitions (cooperativity) modulated by O₂ (homo-activator), H⁺ & 2,3-BPG (hetero-inhibitors); Mb has one site with constant affinity.