Lecture 4 - Functions of Globular Proteins and Enzymes pt 1

• storage of ions and molecules (myoglobin and ferritin)

• transport of ions and molecules (hemoglobin, serotonin transporter)

• defense against pathogens (antibodies, cytokines)

• muscle contraction (actin, myosin)

• biological catalysis (chymotrypsin, lysozyme)

5 functions of globular proteins

ligand (typically a small molecule)

molecule that binds to a protein

binding site

region in the protein where the ligand binds

noncovalent

ligand binds via some _________ forces that dictate protein structure

association rate constant (ka)

kinetics of binding of ligand to protein is described by what constant

dissociation rate constant (kd)

kinetics of unbinding of ligand from protein is described by what constant

the association rate constant and the dissociation rate of constant

when process reaches equilibrium what is equal?

the equilibrium constant (Ka)

the equilibrium composition is characterized by what constant

K_a = [PL] / [P] x [L] = k_a / k_d

equilibrium constant equation

fraction of occupied binding sites

what is θ

θ = [L] / [L] + K_d

equation of θ in terms of equilibrium dissociation constant

θ = fraction of occupied binding sites

what is on the y-axis at K_d intersection?

[P][L] / [PL]

what does K_d equal?

M^-1

association constant units

M

dissociation constant units

K_d < 10 nM (strong)

K_d > 10 uM (weak)

strong and weak binding magnitudes

• size

• shape

• charfe

• hydrophobic/hydrophilic character

what are the binding site and ligand complementary in (creates high specificity)

preformed

“Lock and Key” model assumes that complementary surfaces are ____

induced fit

conformational changes occur upon ligand binding is known as what

tighter binding of the ligand AND high affinity for different ligands

what does induced fit allow for

true

true or false: both the ligand and the protein can change their conformations

• protein side chains lack affinity for O₂

• transition metals would generate free radicals if free in solution and bind O₂

• heme is suitable but Fe²⁺ in free heme would be oxidized to Fe³⁺

• solution → capture the oxygen molecule with heme that is protein bound (hemoglobin, myoglobin)

what is the issue and solution with binding oxygen?

consists of a complex organic ring structure, protoporphyrin IX, with a bound iron atom in its ferrous (Fe²⁺) state

structure of heme

it has a similar size and shape to O₂: it can fit to the same binding site and binds better because it has a filled lone electron pair that can be donated to vacant d-orbitals on the Fe^₂₊ (blocks function of myoglobin, hemoglobin, and mitochondrial cytochromes)

why is carbon monoxide dangerous?

O₂ binds at an angle (also oxygen is hydrogen bonded to distal Histidine) while CO binds in a straight line

difference in angle of binding of O₂and CO to heme group

pO₂ in lungs is about 13 kPa (binds oxygen well) while pO₂ in tissues is about 4 pKa (releases oxygen)

how does hemoglobin know when to bind and release O₂

positive cooperativity

first binding event increases affinity at remaining sites

negative cooperativity

first binding event reduces affinity at remaining sites

concerted cooperativity

all subunits are postulated to be in the same conformation

sequential cooperativity

a conformational change in one subunit makes a similar change in an adjacent unit

allosteric protein

binding of a ligan to one site affects the binding properties of a different site, on the same protein (can be positive or negative)

homotropic

normal ligand of the protein is the allosteric regulator

heterotropic

different ligand affects binding of the normal ligand

positive homotropic regulation

what type of regulation is cooperativity

cooperatively

hemoglobin binds oxygen __________

2 subunits (alpha and beta) both of which are structurally similar to myoglobin

how many subunits is hemoglobin

the sequences are not similar

what is different between hemoglobin and myoglobin

large change at the a₁B₂ contact with several ion pairs broken (goes from tense to relaxed state)

after oxygen binding, what happens to the structure of hemoglobin

T (tense) state

what state is deoxyhemoglobin molecules in

more interactions, more stable, lower affinity for O₂

tense state of hemoglobin

fewer interactions, more flexible, higher affinity for O₂

relaxed state of O₂

T → R conformational change (involved breaking ion pairs between the a₁-B₂ interface)

O₂ binding to hemoglobin triggers what change

T state the iron ball is slightly protruding from heme group

R state the iron ball is in the middle of the heme group

iron ball in middle of heme group difference in T vs R state

Bohr effect

the pH different between lungs and metabolic tissues increased efficiency of the O₂ transport

protons trigger T-state causing release of O₂ molecule

what does increase in protons do to hemoglobin

pH of blood in the lungs is 7.6

pH of blood in the tissues is 7.2

pH of blood in the lungs vs in the tissues

binds to the central cavity of hemoglobin and stabilizes the T state heterotopic regulator) → allows for O₂ release in the tissues and adaption to changed in altitude

what does 2,3-BPG do

Glu→ val in the B chain of hemoglobin → valine side chain and bind to a different hemoglobin molecule to a form a strand which sickles the red blood cells

sickle-cell anemia mutation