Biochemistry (Exam 2)

What are some functions of globular proteins?

• storage of ions and molecules

• transport of ions and molecules

• defensed against pathogens

• muscle contraction

• biological catalysts

What are examples of proteins that store ions and molecules?

• myoglobin

• ferritin

What are examples of proteins that transport ions and molecules?

• hemoglobin

• serotonin transporter

What is are examples of proteins that serve as defense against pathogens?

• antibodies

• cytokines

Ligand

molecule that binds to a protein

Binding Site

a region in the protein where the ligand binds

How can ligand binding be described?

• reversible, transient process of chemical equilibrium

• Protein + Ligand ↔ PL complex

What allows for ligand binding to occur?

noncovalent forces

What allows for protein ligand interaction to be transient?

noncovalent forces

What are the models of substrate binding?

• lock and key

• induced fit

Lock and Key

substrate fits perfectly into the active site

Induced Fit

• ligand binding induces a conformational change that makes interaction more favorable

• can fit in active site, but not perfect

How can protein ligand interactions be described?

quantitatively

How can the kinetics of a protein-ligand interaction be described?

• association rate constant (k_a)

• dissociation rate constant (k_d)

When does the process reach equilibrium?

• when the association and dissociation rates are equal

• k_a[P][L] = k_d[PL]

K_a (equilibrium)

• equilibrium composition

• P + L ↔ PL

• K_a = [PL]/[P][L]

Fraction Bound

what percentage of the protein is bound/the percentage of the protein that is not bound

What does Y depend on?

• free ligand concentration

• K_d

What are the conditions in determining K_d experimentally?

• ligand concentration is known

• ligand increases, and binding is measured

• protein stays the same

How can the k_d be determined?

• graphically

• least-squares regression fit to data

How can interaction strength be expressed?

• K_a

• K_d

• binding free energy ∆G^o (kJ/mol)

K_a (interaction strength)

• binding affinity

• M^-1

• association constant

K_d (interaction strength)

• binding affinity

• dissociation constant

• M^-1

• the point at which half fraction bound

• the tighter the affinity, the lower the number

What is the relationship between the association and dissociation constant?

K_d = 1/K_a

What is the relationship between ∆G and K_d?

the lower the K_d and the lower the G the more favorable binding

What is considered to be strong binding?

K_d < 10nM

What is considered to be weak binding?

K_d > 10 um

How many hemes are in a hemoglobin?

4

What the problems that heme proteins address?

• amino acid side chains lack affinity for O₂

• some transition metals bind O₂ well, but would generate free radicals

• Organometallic compounds are most suitable, but Fe²⁺ in free heme it would be oxidized to Fe³⁺ and not bind O₂

Why are heme proteins useful?

capture the oxygen molecule with heme that is protein bound can maintain ferrous state

O₂ Binding State

• coordinated by Fe of heme and His64

• O₂ coordinated with the distal histidine

• the iron is bound to the proximal histidine

Reversible binding

oxygen is released without the protein being denatured

What are the characteristics of heme as a prosthetic group?

• binds tightly

• required for function

• not reversible

How does oxygen binding change the position of the heme?

when O₂ binds to the iron in heme there is a partial transfer of an electron from Fe²⁺ to oxygen

What does the distal His do?

prevent release of O₂^- and maintains Fe²⁺ heme

What is happening when oxygen binds to myoglobin?

• iron in heme binds to proximal His residue in myoglobin

• distal His helps stabilize O₂ binding

How is the binding of O₂ measured?

pO₂

Myoglobin

• main oxygen storage protein

• stores oxygen in tissues

• monomer

• binds O₂ tightly and has a high occupancy of binding sites tissue and lung

Hemoglobin

• transports oxygen to tissues

• tetramer

• subunits are structurally similar to myoglobin

• 2-α and 2-β monomers are arranged into a tetramer

• each monomer has a heme prosthetic group that can bind O₂

Why is pH important for oxygen binding?

there must be proper protonation of the histidine for this interaction to occur

What does myoglobin’s tight O₂ binding mean?

• myoglobin could bind O₂ in the lungs like hemoglobin but would not release it in the tissues

What does hemoglobin’s affinity change allow for?

• high efficiency of O₂ binding in the lungs and release in tissues

• partial pressure has to drop below normal in order for this exchange to happen

T → R Transition

a physical structure change in hemoglobin that allows transfer from high to low affinity

T State

• predominant in deoxyhemoglobin

• stabilized by multiple ion interactions between monomers α1β2 and α2β1

• more rigid

• less O₂ affinity

• many ionic interactions

What happens when O₂ binds to the T state?

• it induces a conformational change

• the affinity of the 3 binding sites changes

R-State

• more flexible

• higher affinity for oxygen

What is the T → R state transition and example of?

• allostery

• binding of oxygen at one site affects binding at other sites in the protein

What is required for cooperativity?

multiple ligand binding sites

What do you have if you have cooperativity?

allostery

What is an example of cooperativity?

• unbound protein is in a state that has lower affinity for ligand

• binding of ligand to one binding site stabilizes the protein both near the site and more distant

• this increase in stability also increases binding affinity of the second open binding site

What interactions occur when hemoglobin is in the T state?

• His is the C-term residue of the β subunit

• it stabilizes the T-state via two ionic bonds

• R-group interacts with Asp

• COO^- of terminal carboxyl interacts with α chain Lys

• the aspartate OOH interacts with the positively charged histidine

• these interactions break in R

What happens to the helices when oxygen binds?

• helix F is pulled in

• helix C movement changes His interaction

Why does myoglobin have no cooperativity?

there is only 1 binding site for O₂

Why does hemoglobin have cooperativity?

• there are multiple binding sites

Positive Cooperativity

increase in affinity

Negative cooperativity

decrease in affinity

What does the fraction bound equation become when there are multiple binding sites?

Hill Equation

gives an indication of whether or not there is cooperativity

n_H

• slope of the Hill equation line

• measure of cooperativity

• always ≤ n-1

n_H = 1

no cooperativity

n_H < 1

negative cooperativity

n_H > 1

positive cooperativity

What are the final products of glucose metabolism?

• CO₂

• H₂O

Where is CO₂ produced in the body?

tissues as they catabolize glucose

What does the body use to maintain blood pH?

bicarbonate

What causes changes in pH in the blood?

• actively metabolizing tissues generate CO₂ and H⁺

• this lowers the pH of the blood near the tissues

How does pH effect O₂ binding?

• the His and Lys interactions are pH dependent because the salt bridge stabilizes the T state

• at a lower pH the T state is stabilized because there are many protons around, allowing His to stay protonated

What does does hemoglobin affinity depend on?

pH

How can O₂ efficiency between the lungs and tissue be increased?

the Bohr effect

Bohr Effect

hemoglobin’s affinity for O₂ decreases when the concentration of CO₂ increases

How does the Bohr effect apply to hemoglobin in the tissues?

• H⁺ and CO₂ are high tissues in tissues, but O₂ is low

• these factors stabilize the T-state

• decreasing O₂ affinity

• O₂ is released, H⁺ and CO₂ are bound

How does the Bohr effect apply to hemoglobin in the lungs?

• O₂ is high, H⁺ and CO₂ are low

• CO₂ is exhaled shifting bicarbonate equilibrium back

• The R state is favored and O₂ is bound

What does hemoglobin transport?

• O₂

• H⁺

• CO₂

BPG

• modulates O₂ binding to hemoglobin

• binds between β subunits and stabilizes the T-state

• only binds the T-state

What plays an important role in BPG binding?

partial pressure of oxygen

Why does partial pressure play an important role in BPG binding?

• at lower partial pressure, more oxygen needs to be bound

• the body can adjust BPG levels to increase

• allows for increasing tissue O₂ delivery

Heterotropic Allosteric Modulator

molecules that bind and modulate hemoglobin, but are not its substrate

Homotropic Allosteric Modulator

molecules that bind and modulate hemoglobin and are is substrate

What are examples of heterotropic allosteric modulation?

• H⁺

• CO₂

• BPG

What is an example of a homotropic allosteric modulator?

O₂

Allosteric

binding at one site alters binding/activity at other sites

Modulation

conversion between more active and less active

What are the components of an immune cell?

• variable region

• constant region

What happens when an antibody binds to an antigen?

a slight conformational change

What assays take advantage of the specificity of the antibody-antigen interaction?

• ELISA

• Western

ELISA

1. coat surface with sample (antigens)

2. block unoccupied sites with nonspecific protein

3. incubate with primary antibody against specific antigen

4. incubate with secondary antibody, enzyme complex that binds primary antibody

5. formation of colored product indicates presence of specific antigen

Western Blot

proteins separated by size

What effect does CO have on hemoglobin oxygen binding?

• CO binds taking up a spot for O₂ binding, which reduces capacity of hemoglobin for O₂

• CO shifts the affinity to tighter binding

• since CO is not released then the hemoglobin stays in high affinity R state and does not release O₂ in tissues

What effect does CO have on myoglobin binding?

• more CO needs to be present to have an effect

• when O₂ binds, it increases the affinity more than CO

Enzymes

• almost exclusively proteins

• must be folded to be active enzymes

• often require prosthetic groups for catalytic activity

Prosthetic Group

• can be covalently attached

• can be bound by noncovalent interactions

• required for protein to be active

What are examples of prosthetic groups?

• coenzymes

• cofactors

Coenzyme

organic or organometallic molecules

Cofactors

metal ions

Why does a cell need enzymes?

• milder reaction conditions

• greater reaction specificity

• capacity for regulation

• high reaction rates

Why are milder conditions good for enzymes?

• conducive to conditions in cells

• there cannot be large changes in temperature, pH, solvent, or pressure

Why is greater reaction specificity good for enzymes?

• avoids side products

• a single substrate can be converted to multiple products, but for the desired product a specific enzyme must be used

Why is capacity for regulation good for enzymes?

control of biological pathways

Why are higher reaction rates good for enzymes?

• the reaction happens in a biologically useful timeframe

• products may be thermodynamically favorable, but if there is a high activation energy, they may take too long to occur

What do enzymes do to reactions?

• lower activation energy

• stabilize the transition state of the reaction

• increase both forward and reverse reaction rates the same factor