Lecture 5 - Protein Function

What are globular proteins?

Compact, spherical proteins that perform a wide variety of biological functions, including catalysis, transport, storage, signaling, and defense.

What are the major functions of globular proteins?

• Biological catalysis

• Transport

• Storage

• Structure and movement

• Cell signaling

• Immune defense

What is the function of enzymes (globular proteins)?

They catalyze (speed up) biochemical reactions.

Give two examples of catalytic globular proteins.

• Chymotrypsin

• Lysozyme

What is chymotrypsin?

A digestive enzyme that breaks down proteins.

What is lysozyme?

An enzyme that breaks down bacterial cell walls, helping defend against infection.

What is the function of transport proteins?

They transport ions and molecules throughout the body.

Give an example of a transport protein.

Hemoglobin.

What does hemoglobin transport?

Oxygen in red blood cells.

What is the function of storage proteins?

They store important molecules or ions for later use.

Give examples of storage proteins.

• Myoglobin

• Ferritin

What does myoglobin store?

Oxygen in muscle cells.

What does ferritin store?

Iron (Fe²⁺/Fe³⁺).

Which globular proteins are involved in movement?

• Actin

• Myosin

What is the function of actin and myosin?

They work together to produce muscle contraction and cellular movement.

Which globular protein acts as a hormone?

Insulin.

What is insulin's function?

It transmits signals that regulate blood glucose levels.

Which globular proteins help defend against pathogens?

• Antibodies

• Cytokines

What is the function of antibodies?

They recognize and bind foreign pathogens to help eliminate them.

What are cytokines?

Cell signaling proteins that regulate immune responses and communication between immune cells.

What are the six major functional categories of globular proteins?

1. Biological catalysis

2. Transport

3. Storage

4. Structure and movement

5. Signaling

6. Immune defense

What is a ligand?

A molecule that binds specifically to a protein at a binding site.

What is ligand binding?

The specific interaction between a ligand and its protein binding site, allowing the protein to perform its function.

Why are ligand-binding sites specific?

They have a unique shape and chemical properties that allow only certain ligands to bind.

Is ligand binding usually permanent?

No. Ligand binding is usually regulated and reversible.

What determines ligand binding?

It follows the rules of chemistry and moves toward equilibrium.

What is the binding reaction between a protein and ligand?

P + L ⇌ PL

• P = Protein

• L = Ligand

• PL = Protein–Ligand complex

What does the association rate constant (ka) describe?

The rate at which a protein and ligand bind to form the protein–ligand complex.

What does the dissociation rate constant (kd) describe?

The rate at which the protein–ligand complex falls apart into protein and ligand.

Toward what does ligand binding move?

Chemical equilibrium.

What is induced fit?

Ligand binding causes a conformational change in the protein that improves binding or function.

Why is induced fit important?

It increases binding specificity and protein activity.

Can induced fit involve large structural changes?

Yes. Ligand binding can produce dramatic conformational changes.

What is cooperativity?

A conformational change in one subunit of a multisubunit protein affects the binding of ligands to other subunits.

In what type of proteins is cooperativity observed?

Multisubunit (quaternary) proteins, such as hemoglobin.

What causes cooperativity?

Ligand binding to one subunit changes the conformation of neighboring subunits.

What is the dissociation constant (Kd)?

A measure of binding affinity between a protein and its ligand.

What does a low Kd indicate?

Strong binding (high affinity) between the protein and ligand.

What Kd value indicates strong binding?

Kd < 10 nM

What does a high Kd indicate?

Weak binding (low affinity) between the protein and ligand.

What Kd value indicates weak binding?

Kd > 10 μM

What are the major characteristics of ligand binding?

• Specific

• Reversible

• Regulated

• Moves toward equilibrium

• Often involves induced fit

• May show cooperativity in multisubunit proteins

Why does a lower Kd correspond to stronger binding?

Because the protein–ligand complex is less likely to dissociate, indicating higher affinity.

What is the dissociation constant (Kd)?

A measure of how tightly a ligand binds to a protein. It is inversely related to binding affinity.

What does a low Kd indicate?

High binding affinity—the ligand binds tightly and dissociates less easily.

What does a high Kd indicate?

Low binding affinity—the ligand binds weakly and dissociates more easily.

Which protein-ligand pair has the higher affinity?
X: Kd = 2 μM

Y: Kd = 6 μM

• X (Kd = 2 μM) has the higher affinity because it has the lower Kd.

Which ligand dissociates first?
X: Kd = 2 μM

Y: Kd = 6 μM

Y dissociates first because it has the higher Kd (lower affinity).

If two ligands bind the same protein, which one stays bound longer?

The ligand with the lower Kd, because it has a higher affinity.

Complete the relationship:
Kd ↓ = ?

Affinity ↑

(Lower Kd = Higher affinity)

Complete the relationship:
Kd ↑ = ?

Affinity ↓

(Higher Kd = Lower affinity)

What does it mean if a ligand dissociates easily?

It has a high Kd and therefore low binding affinity.

What does it mean if a ligand rarely dissociates?

It has a low Kd and therefore high binding affinity.

Which protein-ligand pair is one of the strongest known biological interactions?

Avidin and biotin.

Why is the avidin-biotin interaction famous?

It has an extremely low Kd, making it one of the strongest noncovalent biological interactions known.

What does the avidin-biotin interaction demonstrate about Kd?

An extremely low Kd corresponds to extremely high binding affinity.

Rank these ligands from strongest to weakest binding.

• Kd = 2 μM

• Kd = 6 μM

• Kd = 50 nM

• Kd = 5 nM

• 5 nM (strongest)

• 50 nM

• 2 μM

• 6 μM (weakest)

How do you compare binding affinities using Kd values?

The smaller the Kd, the stronger the binding.

"Low Kd = Locked Down."

• Low Kd → Ligand stays bound → High affinity

• High Kd → Ligand leaves easily → Low affinity

What is enzyme specificity?

The ability of an enzyme to bind only certain ligands (substrates) because of the complementarity between the binding site and the ligand.

Why are enzymes highly specific?

Their binding sites are complementary to their substrates in:

• Shape

• Size

• Charge

• Hydrophobic/hydrophilic properties

What determines whether a ligand can bind to an enzyme?

The complementarity between the ligand and the enzyme's binding site.

What is the Lock-and-Key Model?

A model stating that the enzyme's active site is pre-formed and fits the substrate exactly like a key fits a lock.

What does the Lock-and-Key Model assume about the enzyme?

The active site is rigid and already complementary to the substrate before binding.

According to the Lock-and-Key Model, what properties must match between enzyme and substrate?

• Shape

• Size

• Charge

• Hydrophobic/hydrophilic character

What is the Induced Fit Model?

A model in which binding of the ligand causes conformational changes in the enzyme, ligand, or both.

What happens during induced fit?

The enzyme changes shape after ligand binding to create a better fit.

Can the ligand also change shape during induced fit?

Yes. Either the enzyme, the ligand, or both may undergo conformational changes.

What advantage does induced fit provide?

It allows tighter binding between the enzyme and substrate.

How does induced fit affect binding affinity?

It increases binding affinity by improving complementarity.

Why is induced fit important for enzymes?

It helps enzymes stabilize the transition state, making reactions occur more easily.

What is the transition state?

A high-energy intermediate formed during a chemical reaction.

How do enzymes speed up reactions using induced fit?

They stabilize the transition state, lowering the activation energy.

Which model better explains enzyme function?

The Induced Fit Model, because enzymes are flexible and often change shape upon substrate binding.

Which model explains stabilization of the transition state?

The Induced Fit Model.

What are the two major models of enzyme specificity?

• Lock-and-Key Model → rigid active site

• Induced Fit Model → flexible active site with conformational change

Why is induced fit considered more accurate than the Lock-and-Key Model?

Because enzymes are dynamic, not rigid, and binding often causes conformational changes that improve substrate binding and catalysis.

Why is oxygen transport necessary in the body?

Oxygen is poorly soluble in aqueous solutions, so simple diffusion is only effective over a few millimeters.

Why can't oxygen rely on diffusion alone?

Because oxygen is poorly soluble in water, making long-distance transport by diffusion impractical.

Which transition metals bind oxygen strongly?

Iron (Fe) and copper (Cu).

Why is free iron dangerous in cells?

Free iron promotes the formation of reactive oxygen species (ROS), which can damage cells.

What is heme?

A protoporphyrin ring that contains an iron (Fe²⁺) atom capable of binding oxygen.

What is the function of the heme group?

It binds oxygen while safely holding iron in the proper oxidation state.

What oxidation state of iron binds oxygen?

Fe²⁺ (ferrous iron).

Which oxidation state of iron cannot bind oxygen effectively?

Fe³⁺ (ferric iron).

How does heme help maintain oxygen-binding ability?

It keeps iron in the Fe²⁺ state, preventing oxidation to Fe³⁺.

Why is the heme group buried inside proteins?

To prevent nonspecific, irreversible binding of oxygen and other molecules.

What is the advantage of burying heme within a protein?

It protects the iron, controls oxygen binding, and reduces unwanted reactions.

Name three proteins that contain heme.

• Hemoglobin

• Myoglobin

• Cytochromes

What is the function of hemoglobin?

Transports oxygen in the blood.

What is the function of myoglobin?

Stores oxygen in muscle tissue.

What is the function of cytochromes?

They participate in the electron transport chain during cellular respiration.

Which gases bind heme with greater affinity than oxygen?

• Carbon monoxide (CO)

• Nitric oxide (NO)

Why is carbon monoxide (CO) highly toxic?

CO binds heme much more tightly than oxygen, preventing oxygen transport.

Why can nitric oxide (NO) affect oxygen transport?

NO also binds strongly to heme iron, competing with oxygen.

What are the major functions of the heme group?

• Safely binds oxygen

• Maintains iron as Fe²⁺

• Prevents formation of reactive oxygen species

• Prevents irreversible oxygen binding

Why is Fe²⁺ required instead of Fe³⁺ for oxygen transport?

Fe²⁺ binds oxygen, whereas Fe³⁺ does not bind oxygen effectively.

Why is carbon monoxide poisoning dangerous?

CO has a much higher affinity for heme than oxygen, preventing oxygen delivery to tissues.

How can oxygen binding to heme be detected?

By UV-Visible (UV-Vis) spectrophotometry.

Why can UV-Vis spectrophotometry detect oxygen binding?

Because the heme group is a strong chromophore that absorbs light in the UV-visible range.

What is a chromophore?

A part of a molecule that absorbs light, producing characteristic absorption peaks.