Chapter 5: Protein Binding, Molecular Rcognition, and Allostery

hemoglobin

• allosteric protein in red blood cells

• tetrameter of 4 polypeptides

• displays cooperative binding and release → good for transport

• like but not an enzyme

how does the tetrameter of hemoglobin differ in deoxy vs oxy state

• deoxyhemoglobin: T state → ab dimers excessively linked, including carboxyl teminus, in deoxyhemoglobin

• oxyhemoglobin: R state → occurs when O2 binds

myoglobin

• single polypeptide

• binds oxygen in muscle cells → no cooperative binding bcz they are for storage not transport

binding curve for myoglobin

• hyperbolic

• half-sat point (P₅₀) of 2 → high oxygen affinity

what does oxygen binding rely on for myoglobin and hemoglobin

oxygen binding is a function of partial pressure of oxygen (PO₂)

• myoglobin has high O2 affinity thus doesnt change much with [O2]

• hemoglobin has low o2 affinity and changes a lot with [o2] (big dif from tissues vs lungs)

secondary structure of myoglobin

lots of alpha helicies linked by turns → compact globular protein

what allows myoglobin and hemoglobin to bind O2

prosthetic group → heme. myoglobin has 1, hemoglobin has 4

heme structure

• central iron atom surrounded by a protoporphyrin (orgnanid) compound

  • photoporphyrin: 4 methine linked pyrrole rings + 4 methyl groups, 2 vinyl groups, 2 propionate chains

  • iron: ferrous form (2+)

• hydrophobic (except the propionate)

heme iron attachment site

• attaches to the 4 N of the pyroles in the photoporphyrin ring

• 5th and 6th coordination sites on either side of the heme plane

heme iron attachment site: Fe fifth coordination site

iron binds imidazole ring of proximal histidine

heme iron attachment site: Fe sixth coordination site

• oxygen binds here → unoccupied for deoxyhemoglobin and deoxymyoglobin

• when O2 binds, Fe electrons rearrage → Fe gets smaller and moves into porphyrin plane

distal histidine

• opposite side of heme than proximal histidine

• directs O2 to globins instead of dangerous ligands (CO)

• prevents Fe2+ oxidation

• limits access to Fe

• stabilizing O2 binding with H bond

constiuents of hemoglobin

• 2 alpha subuntis and 2 beta subunits

• homodimer of heterodimers → made up of 2 αβ dimers

binding curve of hemoglobin

• sigmoidal curve → cooperative binding and release

• P₅₀ → 26 torr, much lower binding affinity than myoglobin

HbA

refers to hemoglobin tetramer

why is it important that hemoglobin partake in cooperative binding

efficient oxygen transporrt

• lungs: HbA is saturated with o2

• tissues: HbA only 30% saturated, releasing ~40% of its O2

binding of o2 at one site of hemoglobin _ binding affinity of the other sites

increases → highest affinity for its 4th O2

how does hemoglobin change when going from T → R (oxygenated)

• iron moved into heme plane, proximal histidine comes with

• ab dimers rotate and form R state

is hemoglobin best described by concerted model or sequential model

neither

• concerted → 3 O2s bound does force the 4th into R state, but if 1 O2 is bound, other sites remain mostly T state

• sequential → even if only ¼ sites are R state, the 1 makes the next o2 bind easier

allosteric effectors

regulatory molecules that change activity of allosteric enzymes

allosteric effectors of hemoglobin

• 2,3-BPG

• protons

• co2

allosteric effectors of hemoglobin: 2,3-BPG

• same [ ] as hemoglobin in the blood

• anionic (- charge)

• binds to hemoglobin and reduces o2 affinity → 66% of o2 relases

• without this, only 8% of o2 would be released in the tissues

how does 2,3 BPG decrease o2 binding affinity

• preferentially binds T state (bcz its positive pocket) and stabilizes it, reducing affinity and releasing o2

• 2,3-BPG must be expelled before T retransitions into R

overall structure of hemoglobin

hydrophobic outside and hydrophillic inside, except for hydophobic pocket for heme

fetal hemoglobin

• 2 alpha units and 2 gamma units

• y similar to b unit, but serine replaces a histidine at the 2,3-BPG binding site → removes 2+ charges (1 from each his) → decreased 2,3-BPG binding → higher o2 affinity than mother hemoglobin

how does the structural difference in fetal hemoglobin work to ensure baby gets o2

fetal hemoglobin o2 affinity is higher→ quickly picks up o2 when mothers hemoglobin releases it

how does temo affcet o2 binding to hemoglobin

increased temp decreases o2 affinity → shifts curve to the right

what is 2,3-BPG the product of

1,3-BPG from glycolysis

bohr effect

heterotropic regulation hemoglobin by H+ and CO2

• o2 affinity decreases as pH decreases from lungs to active muscle (increased H+)

• o2 affintity decreases as [CO2] increases

bohr effect: pH

• high pH: His is deprotonated (uncharged) → no salt bridge forms with aspartate → o2 binding favoured

• lower pH: His protonates → salt bridge with aspartate → T state forms → O2 release

bohr effect: CO2

• CO2 reacts with terminal amino group of T state → forms carbamate groups (- charge)

• carbamate forms salt bridges → stabilizes T state → o2 release

what else does hemoglobin transport other than o2

some H+ and CO2 → after binding at the active cells, they get released at the lungs

only some H and co2 is transported by hemoglobin. how does the rest get to the lungs?

• H and CO2 become bicarbonate (spantaneously or by carbonic anhydrase)

• bicarbonate released to plasma → reverts to H and CO2 at the lungs

• expired in the lungs

sickle cell anemia

• only homozygous ppl are symptomatic

• sensitive, sickled blood cells → erythrocytes rupture → anemia

• clotting → organ damage

sickle cell anemia: which amino residue is affected

• glutamate replaced with valine→ mutated b unit makes HbS

  • valine interacts with other hydrophobic groups when HbS is deoxy → deforms cells and clots

  • reduced solubility of deoxy HbS but not oxy HbS

how does sickle cell affect oxygen affinity and allosteric properties

unaffected

malaria

• parasite disease

• affects red blood cells → anemia, fever, coma, death

• being a carrier of sickle cell causes resistance → more sickle cell in high malaria populations

how does sickle cell resist malaria

HbS is sensitive → ruptures upon parasite infection

fMRI mechanism

• deoxy and oxyhemoglobin have different magnetic properties → arises from O2 moving Fe into porphyrin ring

• can observe brain acitivty → active parts have more oxyhemoglobin

fractional saturation

value between 0 → 1

• 0 → all o2 sites empty

• 1 → all o2 sites full

pO2

expresses partial pressure of o2 in torr→ like [O2]

what does the steepest part of the hemoglobin binding curve represent

change in pO2 from rest to exercise → more o2 offloads during exersion