5: Allostery in Hemoglobin

the binding of a negative allosteric effector to an allosteric enzyme (K-system) that shows positive cooperativity in substrate binding:

causes a shift to the right in the sigmoidal curve of v v. [S]

Monod-Wyman-Changeux (MWC) model

- inhibitor : negative effector

- inhibitor binds preferential to the T form

- T0/R0 equilibrium is shifted towards T0

- # of S binding sits decreases -> T0/R0 is increased

- decrease the affinity of S binding and increase cooperativity

the addition of a negative allosteric effector to an enzyme that acts as a V-system causes the half-maximum velocity (Km) to ________ and Vmax to _______

remain constant

decrease

K systems

- Vmax does not change

- Km changes in the the presence of A and I

V systems

- Km does not change

- Vmax changes in the presence of allosteric effectors

- R and T forms of the enzyme have the same affinities for S, but differ in their affinity for A and I and their catalytic properties

- when [S] > Km

Hemoglobin (Hb)

- blood

- oxygen transport proteins

- tetrameric

- 2 alpha chains of 141 residues and 2 beta chains of 146 residues

myoglobin (Mb)

- muscle

- oxygen-storage proteins

- 153 residues

- contains heme

what binding curves of Hb and Mb show?

- Hb -> SIGMOID shaped O2 binding curves

- Mb -> MICHAELIS-MENTEN type substrate binding curves

how does Mb and Hb bind to Fe2+

- both uses heme (a ring shaped iron containing molecule that is a prosthetic group for various proteins)

- heme -> binds to oxygemn reversibly

how does oxygen bind to Mb and Hb

- iron interacts w/5-6 ligands -> 4 are the N atoms of the porphyrin and the 5th ligand is donated by the imidazole sides chain (His F8)

- His F8 -> on the 6th or "F" helix and the 8th residue in the helix

- when Mb or Hb bind to O2 -> O2 molecules adds to the heme iron as the 6th ligand -> O2 is tilted relative to a perpendicular to the heme plane

O2 binding ______ Mb conformation

ALTERS

- Fe2+ lies slightly above the the plane of heme

- when O2 binds to Fe in heme of Mb, the heme Fe is drawn toward the plane of the porphyrin ring

- O2 brings the Fe2+ more in line with the plane of heme

- Mb -> this small Fe2+ adjustment is minimal consequences

- Hb -> similar change of Fe2+ -> initiates a series of conformational changes that are transmitted to adjacent subunits -> cooperativity

Mb structure

- MONOMERIC heme protein

- polypeptide "cradles" the heme group

- Fe2+ (ferrous iron) binds to O2

- oxidation of Fe2+ -> Fe3+ (ferric iron)

Metmyglobin

- Mb with Fe3+

- DOES NOT BIND TO O2

- Fe3+ is due to oxidation of Fe2+

Hb structure

- α2β2 TETRAMERIC structure

- αβ dimer of Hb w/packing contacts -> α1β1 and α2β2 contacts are important for subunit packing -> helices B, G, H, and GH corner

- sliding contacts α1β2 and α2β1 are made with the other dimer

α1β1 and α2β2 of Hb are _______. they are _________ during oxygenation.

packing contacts

unchanged

does Hb show cooperativity in O2 binding?

- hemoglobin has sigmoid shaped O2 binding curves -> positive cooperativity

- the 4 heme groups are a long way apart -> cooperativity in oxygen binding is not due to direct heme-heme interactions

how does changes in Fe2+ movement due to O2 binding affect Mb and Hb

- Mb -> little consequences

- Hb -> conformational changes in the hemoglobin molecule

conformational changes of Hb due to O2 binding

- in deoxy-Hb the iron atom lies out of the heme plane

- as O2 binds -> Fe2+ moves closer to the plane of the heme

- as Fe2+ moves, it drags HisF8 and F helix with it

- the change is transmitted to the subunit interfaces -> conformational changes leads to rupture of salt bridges

O2 binding to Hb rotates one αβ pair by _____ degrees

15

salt bridges in Hb

- salt bridges stabilize deoxy-Hb (T state)

- salt bridges are present between diff subunits -> 8 salt bridges

- for oxy-Hb (R state) -> salt bridges are broken

- salt bridges and Hbonds involving interactions bt N and C terminal residues in the α chains

- salt and Hbond involving interactions with C terminal residues of β chains

cooperativity of Hb is MWC or KNF model

- BOTH

- O2 binds first to the 2 α subunits

- large conformational change occurs when 2 O2 are bound to Hb -> increases the affinity of the β subunits for oxygen

- deoxy-Hb has low affinity for O2

- heme group of the β subunits are inaccessible to O2

- 1st O2 binds to α subunit

- small changes in tertiary structure of the other α subunit increases its affinity for O2 3 fold (KNF model)

- Hb with 2 O2 bound is in equilibrium w/a form of the protein in which all four subunits are in the R state -> equivalent to T/R transition in MWC model

- large change in quaternary structure

how does cooperative binding of O2 influences Hb function

- Hb must bind O2 in lungs and release it in capillaries

- Hb becomes saturated with O2 in the lungs -> partial pressure is ~ 100 torr

- in capillaries, pO2, is about 40 torr and O2 is released from Hb

- the binding of O2 to Hb is cooperative -> perfect for function

Mb has a ______ affinity for O2 than Hb at all O2 pressures

greater

- since Mb is an oxygen storage protein

Bohr effect in Hb

- the antagonism of O2 binding by H+ is termed the Bohr effect

- binding of H+ diminishes O2 binding and vice versa

- protonation of His146 induces R-to-T transition of Hb -> decreasing affinity for O2

- in deoxy-Hb -> 3 amino acids residues form two salt bridges -> stabilize the T quaternary structure

in condition of alkalosis, the affinity of O2 for hemoglobin is _______. and the curves shifts _______.

higher

left

CO2 affect in O2 binding of Hb

- Hydration of CO2 in tissues and extremities leads to proton production -> O2 binding decrease

- CO2 + H2O <-> H+ + HCO3-

- these protons are taken up by Hb as O2 dissociates

- reverse occurs in the lungs

- CO2 DECREASES O2 BINDING

bicarbonate dehydration in Hb

- at lung-artery interface

- bicarbonate dehydration (required for CO2 exhalation) consumes extra H+

- promotes CO2 release and O2 binding

CO2 ______ deoxy-Hb

STABILIZES

- stabilizes deoxy-Hb from reacting w/terminal amino groups -> forms a carbamate group (neg charges) and participates in salt bridges

- deoxy-Hb (T state) is stabilizes

2,3 biphosphoglycerate is an _______ effector of Hb

NEGATIVE ALLOSTERIC

- in absences of 2,3 BPG, O2 binding to Hb follows a rectangular hyperbola -> Michealis-menten

- sigmoid binding curve is only observes in presence of of 2,3BPG

- 2,3 BPG binds at a site distant from the Fe where oxygen binds

- 2,3BPG is in the center of a cavity bt two β subunits

- 2,3BPG binding stabilizes the deoxy form (T state) -> reduce affinity for O2

what are the 3 positive changes that the negative charges interact with in the cavity where 2,3BPG is

2 Lys, 4 His, 2 N- termini

fetal Hb

- fetal Hb has a higher affinity for O2

- circulatory system is entirely independent form its mothers when in the womb

- gas exchange is across the placenta

- fetal Hb -> γ chains in place of β-chains -> α2γ2 structure

- lower affinitiy for 2,3BPG -> higher affinity for O2

- γ-chains have a Ser instead of His at position 143 -> lacks two of the positive charges in BPG-binding cavity -> BPG binds less tightly -> fetal Hb thus looks more like Mb in its O2 binding behavior

Hb is a _______ with ______ substrate binding and ______ effectors.

tetramer

cooperative

allosteric

SHOWS FEATURES OF BOTH MODEL