Exam 3 Total Slides

What are the key principles of protein-ligand interactions?

1. Proteins interact with ligands at binding sites that are complementary to the ligand

2. Proteins have separate binding sites for several different ligands

3. Proteins are flexible and that flexibility may play a role in binding to ligands

4. Interactions between ligands and proteins may be regulated

5. In a multisubunit protein, a conformational change in one subunit often affects the conformation of other subunits

What are allosteric proteins (ex: hemoglobin)?

Binding of a ligand to one site affects the binding properties of another site on the same protein

What are modulators?

Ligands that bind to an allosteric protein to induce a conformational change

What is homotropic vs heterotropic?

Homotropic: Normal ligand and modulators are identical

Heterotropic: Modulator is a molecule other than the normal ligand

Memorize these (can skip)

What is the hill equation?

What is hill plot?

plot of log [Y/{1-Y)] versus log [L]

What is hill coefficient?

n_H = slope of a Hill plot

If n_{H > 1} = ?

If n_H < 1 = ?

If n_H = 1 = ?

• If nH = 1, ligand binding is not cooperative

• nH > 1 indicates positive cooperativity

• nH < 1 indicates negative cooperativity

What is the MWC (Concerted) Model and Sequential Model?

The MWC model (Monod–Wyman–Changeux, also called the concerted model) and the sequential model are two theories explaining how ligands bind cooperatively to multi-subunit proteins like hemoglobin.

What is the MWC (concerted) model?

All subunits in a protein are symmetrically related and must be in the same conformation at any given time - either all in the T (tense) state or all in the R (relaxed) state

What is the MWC (concerted) model states?

T state: Lower ligand affinity, favored at low ligand concentration

R state: Higher ligand affinity, favored at high ligand concentration

In the MWC Model, ligand binds more tightly to the?

R state

What is the sequential model?

Each subunit can independently exist in either the T or R state, with no requirement for all subunits to be in the same conformation

In sequential model, equilibrium is altered when?

Equilibrium is altered when additional ligands are bound, progressively favoring the R state

What are the two end products of cellular respiration that hemoglobin carries?

H+ and CO₂

What is carbonic anhydrase?

An enzyme that catalyzes the hydration of CO2 to bicarbonate: CO2 + H2O ⇌ H+ + HCO3-

What are the structural effects of H+ and CO2 binding to hemoglobin?

It favors the T state (it is inversely related to the binding of O2)

What does lower ligand affinity mean? Vise versa

Low: ligand binds to its target molecule with weak attraction, resulting in transient and easily reversible interactions

High: Ligand binds to its target molecule with strong, tight, and more stable attraction and binding.

What is Kd (dissociation constant)?

A measure of the affinity between a ligand and its binding partner

What does a low Kd mean?

A high ligand affinity - ligand binds tightly to the receptor/protein

What does a high Kd mean?

A low ligand affinity - ligand binds less tightly to the receptor/protein

What is the relationship between Kd and affinity?

They are inversely related: lower Kd = higher affinity, higher Kd = lower affinity

Which has a higher affinity: a ligand with Kd = 1nM or 100 nM?

The ligand with Kd = 1 nM

Why is a lower Kd associated with stronger binding?

A lower ligand concentration is needed to achieve substantial binding, indicating tighter binding under the model

What is the difference between the T state vs R state?

T state: Tense

• low O2 affinity

• Favors O2 release

• Stabilized by H+ and CO2

R state: Relaxed

• High O2 affinity

• Favors O2 binding

• Stabilized by O2

High O2 affinity means?

Low O2 affinity means?

High O2

• Hemoglobin binds O2 tightly

• Released H+

Low O2

• Hemoglobin binds O2 weakly

• Binds H+

What does the Bohr effect help us find?

Describes the effect of pH and [CO2] on the binding and release of O2 by hemoglobin

What is the Bohr effect?

↑ CO₂ + ↑ H⁺ → ↓ pH → T state → ↓ O₂ affinity → ↑ O₂ release

Increase of 2,3-BPG affects hemoglobin how?

Hb holds O2 less tightly

More O2 reaches tissues

Decrease of 2,3-BPG affects hemoglobin how?

Hb holds onto O2 more tightly

Less O2 reaches tissues

Where does BPG bind to deoxyhemoglobin?

BPG binds to the cavity between the B subunits in the T state

What does BPG stabilize?

The T state by binding between the positively charged residue cavity

Oxygen binding to hemoglobin is regulated by?

2,3-biphosphoglycerate

2,3-bisphosphoglycerate (BPG) binds to what site?

A binding site distant from O₂-binding site

When does BPG impact vs not impact O₂ affinity at pO₂ ?

• Has little effect on O₂ affinity at pO₂ present in lungs

• Significant impact on O₂ affinity at pO₂ present in peripheral tissues

What is hypoxia?

Lowered oxygenation of peripheral tissues

How does hypoxia relate to BPG?

It causes an increase in BPG

What is the cavity between the β subunits lined with?

Positively charged residues

What are the characteristics for fetal hemoglobin?

• Lower affinity for BPG than normal adult hemoglobin

• Higher affinity for O₂ than normal adult hemoglobin

• Fetus synthesizes a₂ ½ hemoglobin

What is sickle cell anemia?

A homozygous condition caused by a single amino acid substitution, where glutamic acid at position 6 is replaced by valine on the β-globin chains. This mutation creates a hydrophobic patch on the surface of hemoglobin molecules, leading to the characteristic sickling of red blood cells.

What happens between normal and sickle cell hemoglobin when deoxygenated?

HbS becomes insoluble and forms polymers that aggregate

Normal Hb remains soluble upon deoxygenation

True or False: Spontaneous means fast

False; spontaneous refers to something occurring naturally without external influence, not necessarily quickly.

What’s the rate limiting step ofany reaction?

The rate-limiting step of a reaction is the slowest step that determines the overall reaction rate.

How do we get more molecules to transition state and faster?

By:

• increasing the temperature

• using a catalyst

• raising the concentration of reactants

What is the difference between lower activation energy and higher activation energy?

• Lower activation energy means that less energy is required for reactants to reach the transition state, making the reaction faster.

• Higher activation energy requires more energy for the reaction to proceed, resulting in a slower reaction.

What are the six characteristics of enzymes?

1. Required in small amounts

2. Not permanently altered during reaction

3. Cannot affect the thermodynamics of reactions, only the rates

4. Highly specific for their substrates

5. Produce only specific products

6. Can be regulated to meet the needs of a cell

True or False: Enzymes are almost always proteins

True. Most enzymes are proteins but ribozymes are the exception

Enzymes can be conjugated with?

Cofactors and coenzymes

Cofactors vs Coenzymes

• Cofactors are inorganic enzyme conjugates that assist enzymes in their activity

• Coenzymes are organic enzyme conjugates that temporarily bind to an enzyme to help catalyze a reaction.

What is the prosthetic group?

Coenzyme or metal ion that is very tightly or covalently bound to the enzyme protein

What is holoenzyme?

The complete, functional enzyme formed when the inactive apoenzyme binds with its required cofactors or coenzymes

What is apoenzyme or apoprotein?

The protein component of a holoenzyme, representing the enzyme in its inactive form without cofactors or coenzymes

Why would the protein not be active?

Why would the protein be active?

• When the cofactor is not attached to the protein site, the protein is not active

• When the cofactor binds to the protein’s binding site, activating the protein

Where do enzyme-catalyzed reactions take place?

Within the active site

What is the active site and its interactions?

Region of enzyme where substrate binds; usually noncovalent interactions, but sometimes transient covalent bonds form

What is the substrate?

The molecule that is bound to the active site and acted upon by the enzyme to yield a chemical reaction

What is activation energy?

The small energy input required for any chemical transformation which slows the process of thermodynamically unstable reactants

When is something in the transition state?

When reactant molecules reach the peak of the EA barrier

How does the activation energy work as a barrier to slow the process of thermodynamically unstable reactants?

It represents the energy barrier that must be overcome for a reaction to occur, preventing reactants from spontaneously converting into products

What is ground state?

Starting point for either the forward or reverse reaction

What is biochemical standard free-energy change?

∆G′°: the standard free-energy change at pH 7.0

How do enzymes relate to activation energy?

They lower the activation energy required for a reaction, allowing reactants to convert to products more quickly without altering the overall free-energy change of the reaction.

What happens if there is no enzyme in the activation energy?

The reaction would proceed much more slowly because the reactants would need to overcome a higher activation energy barrier on their own.

True or False: Enzymes affect equilibria not reaction rates

False; enzymes speed up the rate of a reaction but do not change the equilibrium position.

True or False: Enzyme catalyst reactions are reversible under right conditions

True; under the appropriate conditions, enzyme-catalyzed reactions can proceed in either direction, allowing the reaction to reach equilibrium.

What is the rate-limiting step?

The step in a reaction with the highest activation energy and he slowest step that determines the overall reaction rate.

Reaction equilibria are linked to?

Reaction rates are linked to?

Reaction equilibria are linked to the standard free-energy change for the reaction, ∆G′

Reaction rates are linked to the activation energy,∆G⁺⁺

What are the 7 classes of enzymes?

1. Oxidoreductases

2. transferases

3. Hydrolases

4. Lyases

5. Isomerases

6. Ligases

7. Translocases

What is oxidoreductases?

Enzymes that catalyze oxidation-reduction reactions, transferring electrons (hydride ions or H atoms)

• Oxidation/reduction

What is transferases?

Enzymes that catalyze the transfer of functional groups, such as methyl or phosphate groups, from one molecule to another.

• Transfers a group from one molecule to another

What is hydrolases?

Enzymes that catalyze the hydrolysis of various bonds, such as peptide, ester, or glycosidic bonds, by adding water.

• Break bonds using water

What is lyases?

Enzymes that catalyze the formation of C—C, C—O, C—N, or other bonds by elimination, leaving double bonds or rings, or addition of groups to double bonds

• Break/form bonds without hydrolysis

What is isomerases?

Enzymes that catalyze the transfer of groups within molecules, converting it into its isomeric form without adding or removing atoms.

• Rearranges a molecule

What is ligases?

Enzymes that catalyze the formation of C—C, C—S, C—O, and C—N bonds by condensation reactions coupled to cleavage of ATP or similar cofactor

• joins molecules together, usually using ATP

What is translocases?

Enzymes that catalyze the movement of ions or molecules across membranes or their separation within membranes.

• Moves substances across membranes

What are isozymes?

Different structural forms of enzyme that does same reaction

How do you read enzyme numbers?

1. Class

2. Reaction

3. Substrate type

4. Specific substrate

If an enzyme oxidizes things by reducing NAD(P)+, what is it called?

Dehydrogenases

True or False in hydrolases, substrate is some form of amide or ester

True. Hydrolases act on substrates that are typically amides, esters, or other similar bonds, breaking them down through hydrolysis.

Synthases vs Synthetases

Synthases: Usually lyases and involved in creating C—C bonds

Synthetases: Usually ligases and use NTPs and release energy

• lyases: cleave bonds

• ligases: create/bond molecules together

What is mutase?

An enzyme that catalyzes the transfer of a phosphate group from one position to another within the same molecule.

Translocase is the movement of molecules or ions across membranes, what does it form or what are its characteristics?

• Create gradients

• Pump proteins into organelles or out of cells

• Catalyze other motions

• Changes chemical environment/location of molecule

What makes up the enzyme substrate (ES) complex?

Enzyme + substrate

True or False: The active site contains permanent covalent bonds?

False; the active site forms temporary interactions with the substrate, not permanent covalent bonds.

The interactions that the active site performs involve?

Cofactors and coenzymes that assist enzyme function

What are co-substrates?

When coenzymes exit the reaction altered from their original form due to a loose binding of coenzyme and enzyme

• Ex: NAD+ reduced to NADH + H⁺

How are coenzymes attached to the enzyme?

Either loosely bound (co-substrates) or tightly bound (prosthetic groups) to the enzyme

What are prosthetic groups derived from?

Derived from vitamins

When is ATP considered a coenzyme/co-substrate?

When used only for energy

What is NAD (nicotinamide adenine dinucleotide)

• Coenzyme involved in redox reactions

• Soluble electron carrier

• 5’-5’ phosphate linkage

What is FMN & FAD?

• Flavin coenzymes

• Derived from riboflavin (vitamin B2)

• Tightly bound or covalently attached to enzymes (prosthetic groups)

• Involved in:

• Electron transport chain

• Saturation of bonds

• Not free in solution because they need to stay localized/bound to the enzyme

NADH is?

Nicotinamide, the electron acceptor which accepts a hydride ion (H-) becoming NADH

NADH must be what in order to go back to NAD+? This causes ?

NADH must be oxidized back to NAD+ before NAD+ can accept another hydride

What is dehydrogenases?

Enzymes that facilitate the removal of hydrogen atoms from a substrate, typically transferring them to electron carriers like NAD+ or FAD.

What is oxidase?

Enzymes that help a molecule lose electrons by transferring them to oxygen, usually creating water or hydrogen peroxide but oxygen does not appear in the oxidized product

What is mixed function oxidases?

Enzymes that catalyze reactions where one substrate is oxidized while another is reduced, often adding one oxygen atom to the substrate and turning the other oxygen atom into water.

What is oxygenases?

Enzymes that incorporate one or more atoms of oxygen directly into the substrate molecule during a chemical reaction and oxygen does appear in the oxidized product

Monooxygenases vs Dioxygenases

Monooxygenases: One oxygen atom is incorporated in the product

Dioxygenases: Two oxygen atoms are incorporated in the product

What is pyridoxal phosphate (PLP)?

• Coenzyme involved in transamination

• Helps transfer amino groups between molecules

• PLP attached to amino acids and becomes pyridoxamine