biochem lecture 8: protein function

1. ligand? alternate name

2. binding site? 2 alternate names

1. small mC that binds to a protein

2. substrate

3. protein region where ligand binds

4. active/catalytic site

3 aspects of ligand binding

1. what are they

2. why are they important for life.

3. What causes ligand binding to be T.?

1. specificity

   1. allows specific functions

2. reversible = A+B <-> AB

   1. allow cells to be dynamic

3. transient = short-lived bc ligand binds via same noncovalent forces as sec/tert protein structure

   1. allows cells to be dynamic

the reversible/transient protein-ligand chemical reaction

L + P <-> LP

1. Kd: what is it

2. theta: what is it

3. [P]: what is it

4. [L]: what is it

5. [PL]: what is it

1. what are the units?

1. Dissociation Constant = ratio of (dissociated protein) + (dissociated ligand) / (protein/ligand complex)

2. theta = point at which 50% (.5) of ligand is bound to protein

3. [P] = concentration of dissociated protein

4. [L] = concentration of dissociated ligand

5. [PL] = concentration associated protein/ligand complex

1. units except for theta are M (molar)

2. theta expressed as a %

1. Kd = ? = ?

2. Ka = ?

3. theta = ? = ?

4. Ltotal = ?

   1. what about normally?

5. Ptotal = ?

1. Kd = ([P][L]) / [PL] = 1/Ka

2. Ka = 1/Kd

3. theta = [L] / ([L]+Kd) = [PL]/[Ptotal] = 50%

4. [Ltotal] = [PL] + [L]

   1. when [L] » [P] (normally), [Ltotal] = [L]

5. [Ptotal] = [PL] + [P]

smaller Kd affect on affinity

higher affinity = tighter binding

Ligand binding graphical analysis:

1. what equation makes the curve

2. where is theta, Kd, and [L]

1. theta = [L] / ([L] + Kd])

Ligand binding graphical analysis:

1. what variable determines the shape of the curve?

2. what is the relationship between that variable, the shape, and a proteins binding affinity?

3. how does a higher kd or a higher affinity protein look on the graph

1. Kd determines the shape

2. smaller Kd = steeper line = higher affinity (higher [L] needed for theta = 50%)

3. higher Kd = more horizontal line = lower affinity

when theta = ½ or 50%, what are Kd and [L]

when theta = 50%, Kd = [L]

Kd is the ligand concentration when ____

half the ligand is bound (theta = 50%)

1. why don’t we use proteins, transition metals, or organometallic compounds like free heme as oxygen binders?

2. what is the body’s solution?

1a. protein side chains lack O2 affinity

b. transition metals can bind O2 but make free radicals

c. organometallics like free heme can bind O2 but can be oxidized into Fe3+

1a. O2 mC captured w/ protein-bound heme = hemoglobin

hemoglobin v myoglobin:

1. what purposes?

2. what Kd and why

1. myoglobin = O2 storage protein = low Kd to bind tight

2. hemoglobin = O2 transport = low AND high Kd depending on environment

1. what type of protein is heme?

2. 2 part structure?

3. how many valences and what do they bind in myoglobin?

1. metalloprotein

2. Fe2+ contained in center of 4 planar pyrrole ring groups

3. 2 valances open in free heme: 1 binds F helix Histidine, one binds Oxygen

4 parts of Myoglobin and how they bond?

1. His of F helix bonds one of the valences of Fe2+ in heme

2. other Fe2+ valence bonds O2

3. His of E helix bonds w/ the same O2

2 reasons carbon monoxide poisoning happens

1. CO similar size/shape to O2, but binds Fe2+ valence in heme w/ much stronger affinity

2. competes w/ O2 and blocks hemoglobin, myoglobin, and mitochondria

oxygen binding graphical analysis:

1. what equation governs it?

2. where are the 3 variables? units?

3. how do they relate to the ligand binding graph variables?

4. why is myoglobin a good storage molecule

1. theta = pO2 / (pO2 + P50)

2. P50 is like Kd but a pressure bc O2 is gas = kPa

3. pO2 is like [L] = kPa

4. low P50 so high affinity = won’t give it up

hemoglobin v. myoglobin structure:

1. why can hemoglobin bind 4 O2 Mcs?

1. hemoglobin tetramer 4 subunits

2. each subunit looks similar to myoglobin

3. myoglobin can bind 1 O2 (other valence binds His)

4. hemoglobin has 4 subunits, so it can bind 4

1. sigmoidal binding curve = ?

2. why is hemoglobins curve sigmoidal

1. sigmoidal = switch = cooperativity (whether positive or negative)

2. O2 binding causes switch between 2 states

   1. high affinity state (for O2 uptake)

   2. low affinity state (for O2 release)

1. cooperativity

2. allostery

3. which protein exhibits both?

1. binding of one ligand allows others to bind

2. ligand binding either helps another ligand bind or inhibits another from binding

3. hemoglobin

   1. O2 binding → T and R states

lungs =

tissue =

lungs = load = high affinity

tissue = let go = low affinity

T (Tense) hemoglobin v. R (Relaxed) hemoglobin

1. # stabilizing ion pairs

2. HisHC3 ion-paired (protonated)

3. Fe in or out-of-plane

4. affinity of O2

5. deoxy or oxyhemoglobin

1. T more stable / ion pairs

2. T protonated

3. T out of plane

4. T low affinity

5. T deoxy

1. what reaction is responsible for T state?

2. what is normally responsible for R state?

3. what reaction goes from T to R?

   1. cooperatively/allostery?

1. protonated His HC3 moves Fe2+ out of plane and this conformation messes O2 binding up

2. in phase Fe stabilizes R state

3. O2 binds one subunit → allotteric conformation changes in plane→ Fe more likely to bind more O2’s

   1. cooperative binding / positive allostery

Hill Plot:

1. what does it help us do?

2. What is nH?

3. How is nH expressed on the graph?

4. what does hemoglobin’s plot look like?

5. how does nH value affect hemoglobin cooperativity and structure/state?

1. tells us whether there is + or - cooperatively?

2. Hill coefficient = measures cooperativity

3. nH is the slope of the protein’s plot

4. sigmoidal

5. nH > 1 = cooperativity = O2 binding changes structure to allow more binding = R state

6. nH = 1 = no cooperativity = T state

7. nH < 1 = negative cooperativity = wrong mC binding changes structure to inhibit more binding

1. what causes hemoglobin hill plot shape

2. what causes nH (slope) increase on hemoglobin hill plot?

1. sigmoidal binding curve

2. changes between high affinity and low affinity states

1. 2 steps how does CO2 modulate Hb affinity for O2?

2. how does CO2 amount affect:

   1. acidity

   2. O2 binding curve

   3. more/less tense?

1. CO2 ionizes in water to give protons = more acidic

2. H+ transport by protonated His HC3 stabilizes T state

1. more CO2 = more acidic = shift to the right = more tense

2. Less CO2 = less acidic = shift to the left = less tense

1. Bohr effect

2. Bohr state?

1. changes in blood acidity and CO2 concentration affect Hb’s ability to bind/release O2

2. when Hb’s His HC3 is protonated by CO2 protons + salt bridges from - carbamate = tense state

2(3) ways CO2 effects Bohr effect/tense state

1. ionizes in water → HisCH3 protonation → T state

2. carbamate bonding releases a proton → more HisCH3 protonation → T

3. (-) carbamate forms salt brdges → T state

2,3 bisphosphoglycerate (BPG):

1. what does it do to Hb?

2. effect on binding curve?

3. role in altitude acclimatization

1. ion pair binds and stabilizes T state

2. shifts to the right

3. body produces BPG to facilitate O2 release in low oxygen environment

sickle cell homozygous v. heterozygous

1. homo = AA mutation causes subunit interactions, forms long fibers that deform cell shape and clog capillaries

2. heterozygous “sickle cell trait” = may confer malaria resistance

Fetal Hb:

what does it need and effect on curve

1. needs more O2

2. shifts to the right

6 things that shift O2 binding curve to the left

high pH (more basic) = less CO2

low temp

low 2,3-BPG

methemoglobinemia

carboxyhemogolbinemia

high O2 affinity hb variants = Fetal Hb

4 things that shift O2 binding curve to the right

low pH (more acidic) = more CO2

high temp

high 2,3 BPG

low O2 affinity Hb variants