biochem 1 - E2

ligand

small molecule that binds to a protein

Lock-and-Key Model

where the binding site and ligand have “perfect” complementary structures for each other [protein DOES NOT change shape]

Induced Fit Model

where the ligand causes the protein to change shape, producing a tighter fit (this allows for higher affinity)

Protein Specificity

ability of a protein to selectively bind ligands based on size, shape, charge, and hydrophobic/hydrophilic properties

Noncovalent Interactions

Weak interactions (hydrogen bonds, ionic interactions, hydrophobic interactions, van der Waals forces) that mediate ligand binding

Define K_d constant.

is the dissociation/equilibrium constate where the concentration of ligand at which half of a partner’s binding sites are occupied when at equilibrium

K_d equation

K_d = [P] [L]/ [PL]

Fractional Saturation (θ)

fraction of total binding sites occupied by ligand (has realationship to Kd)

(θ) equation

(θ) = occupied binding sites / total binding sites

(θ) equation in terms of Kd

(θ) = [L] / [L] + Kd

On a graph, if (θ) is known (like 0.5) then ______.

Kd will equal [L] where (θ) (y-axis) is equal to 0.5

A high (increased) K_d implies?

weaker affinity due to increased dissociation (inverse relationship)

A low (decreased) K_d implies?

higher affinity due to decreased dissociation (meaning its harder for it to divide into P and L)

Rank the following based on binding affinity (or K_d) [highest to lowest]

10 picoM > 10 nanoM > 10 microM > 10 miliM [smallest value = highest affinity]

For a K_d graph, curving to the right demonstrates ______?

→ up to the right = increased K_d (lower affinity)

For a K_d graph, curving to the left demonstrates _____?

← down to the left = decreased K_d (higher affinity)

For the graph, what is the K_d for proteins X and Y?

X → 2 and Y → 6. X has a higher affinity!

Orthosteric Binding

binding of a ligand at the protein's active site or natural ligand-binding site

Allosteric Binding

binding of a ligand at a site different from the active site, causing a functional change in the protein

Positive Cooperativity (Allosteric Regulation)

binding of one molecule increases affinity for future ligand binding (LIKE Hb)

Negative Cooperativity (Allosteric Regulation)

binding of one molecule decreases affinity for future ligand binding

In relation to gases, like O₂, what equation relates [L] and K_d.

[L] is replaced by partial pressure of O2 and Kd is replaced by half PP of O2..

Define heme.

Protoporphyrin ring containing Fe²⁺ that binds oxygen (found in both hemoglobin and myoglobin)

Hemoglobin (Hb)

Tetrameric protein in red blood cells composed of 2 α and 2 β subunits that transports oxygen.

Max oxygen capacity of Hb?

1 hemoglobin molecule can bind four oxygen molecules (recall car w/ 4 passengers)

How does Hb aid in oxygen transport?

it loads oxygen in the lungs and releases oxygen to body tissues

Hb Loading and Release Cycle

high oxygen partial pressure in lungs favors oxygen binding; lower oxygen partial pressure in tissues favors oxygen release

When oxygen binds to Hb, what Hb confirmations are possible?

T and R states

T State (Tense State)

kPa is higher, so low-affinity conformation favored when oxygen concentration is low (predominant form of deoxyhemoglobin) [on graph, most bottom curve]

R State (Relaxed State)

kPa is super low, so high-affinity conformation favored as oxygen concentration increase [on graph, curve ‘hugs’ Y-axis]

Describe T → R Transition

structural change that occurs when oxygen binds hemoglobin

What measure cooperativity? (ex. like in Hb)

Hills coefficient (n)

When Hill Coefficient (n) = 1, this implies?

there is no cooperativity present

When Hill Coefficient (n) > 1, this implies?

positive cooperativity (hemoglobin exhibits this behavior!)

When Hill coefficiantf (n) < 1, is implies?

negative cooperativity

MWC (Concerted Model)

entire hemoglobin molecule exists as either all T state or all R state

KNF (Sequential Model)

individual subunits switch conformation independently, allowing T and R states to coexist

Regulators for oxygen binding to Hb?

1. pH

2. CO₂

3. 2,3-BPG

How does pH affect O2 affinity to Hb?

low pH = shifts oxygen-binding curve right and decreases oxygen affinity (AKA Bohr effect)

high pH = Increases oxygen affinity

How does CO2 effect?

CO₂ promotes carbamate formation and protonation, stabilizing the T state and decreasing oxygen affinity

Define 2,3-BPG.

Negatively charged molecule in red blood cells that binds hemoglobin and decreases oxygen affinity

What is 2,3-BPG’s affect on O2 affinity?

Stabilizes the T state and shifts the binding curve to the right + decreased amount = increased affinity

Fetal Hemoglobin (HbF)

Hb variant with higher oxygen affinity than adult hemoglobin, allowing oxygen transfer from mother to fetus.

Define myoglobin.

Monomeric oxygen-storage protein found in muscle tissue

Carbon Monoxide Toxicity

CO has a much higher affinity for free heme than oxygen and can interfere with oxygen storage

Role of distal Histidine (His) for decreasing CO binding?

Amino acid residue near the binding site that forces CO to bind at an angle, reducing its affinity for the iron center. Reduces carbon monoxide binding and helps preserve oxygen storage function.

Determining Kd from a Binding Curve

Find the ligand concentration where θ (fractional saturation) equals 0.5

Smaller Kd Between Two Proteins

The protein with the smaller Kd has the higher ligand affinity

Protein Effect on CO Binding

The protein environment decreases the CO/O₂ binding ratio compared with free heme

Why do proteins Reduce CO Binding? (like myoglobin)

oxygen is stabilized by hydrogen bonding with histidine, whereas CO geometry prevents optimal hydrogen bond formation

rue Statements About Myoglobin and Hemoglobin

Both contain heme; myoglobin binds oxygen more tightly; tertiary structures are similar; functions differ (storage vs transport)