Biochemistry Exam #2, Chapter 4: Protein and Enzyme Mechanisms

Roles of both myoglobin and hemoglobin

oxygen transport and have a heme prosthetic group

Physiological Role of Myoglobin

Transports O2 in rapidly respiring muscle, monomer, store oxygen in muscle and has a high affinity for oxygen, diving animals have large concentration of myoglobin to keep oxygen supplied to muscles

Physiological Roles of Hemoglobin

Found in red blood cells, carries oxygen from lungs to tissues and removes CO2 and H+ from blood to lungs, lower affinity for oxygen than myoglobin and tetramere, with two sets of similar units (a and b)

Prosthetic group

a molecule bind tightly to a protein and is essential for its function

Features of Fe 2+ porphyrin complex

protoporphyrin ring with a Fe2+, oxygen can only bind in the +2 state, binding prevents Fe2+ from being oxidized

How many coordination bonds does Fe 2+ have

six, four of these are N atom of the porphyrin and the 5th and 6th are the proximal and distal histidine residues. Presence of O2 is required for coordination through the distal hisitidine

Without oxygen binding what is shape of ring

With Fe2+ being too big for the ring it starts to pucker

How does O2 binding help with puckering

pulls electrons from Fe2+ allowing it to fit into the ring, flattening it out pulling F helix.

Ligand

Generally speaking a ligand is a molecule that binds to another molecule

Reversibility

Reversible binding a common feature fo many biomolecules

How reversibility affects function of oxygen and heme iron

it is reversible because the protein structure changes under different conditions and results in altered affinity for the oxygen ligand, the binding stabilizes the protein and changes orientation of F helix

T (tense) State

oxygen unbound

R (relaxed) State

bound oxygen

What rotation movement binding induces

15 degree of a B heterodimer

How small structure changes result in large changes for whole protein in hemoglobin

rotation between the T and R state can alter as many as 50 noncovalent interaction at the interface between alpha Beta dimer, can change affinity of oxygen

What of the subunits is most difficult to bind in hemoglobin

The first one is the hardest, as we bind each it gets easier.

Allostery

Interactions that occur within a protein between spatially distant sites

Allosteric behavior of hemoglobin

allosteric interaction affect oxygen binding, R has higher state has increased affinity than T

Cooperativity

binding of the first ligand to the protein complex facilitates binding of additional ligands on the same protein, involves multiple subunits each with a binding/active site

Positive cooperativity

ligand binding increases the affinity for other sites, making it easier for other ligands to bind to the protein

Negative cooperativity

Reduced affinity

Sequential model

a two state model of cooperative binding behavior in a tetrameric protein complex in which binding of ligand to one subunit causes that subunit to switch to a new state

Concerted model

model of cooperative binding behavior in a protein complex which the proability of being in one of two states fo complex affected by ligand binding (all or nothing), no intermediates.

Three key features of ligand protein interaction

Ligand binding is a reversible process involving noncovalent interaction, induces/stabilizes structural conformations in target proteins and the equilibrium between ligand bound proteins and ligand free protein can be altered by binding of effector molecules, which induce conformational changes in the protein that increase or decrease ligand affinity

Effector molceules that affect oxygen affinity

O2, CO2, H+ 2,3-BPG

Why CO is toxic to animals

high binding affinity, R state stabilizaiton, complex formation and peripheral oxygen deprivation

equation for fraction of protein binding sites occupied

Occupied binding sites / Total binding sites

The oxygen binding curve for myoglobin

Has hyperbolic shape, resting tissue O2 levels are high, oxygen strorage molecule

Oxygen binding curve for hemoglobin

sigmoidal shape indicated cooperative binding = allosteric effect, high pO2 in lungs and hemoglobin almost fully bound to O2, lower pO2 in tissues results in release of O2 from hemoglobin

Positive allosteric effectors

increased affinity

Homotropic allosteric effector

same thing affecting oxygen binding

How does pH decreases affect association of O2

dissociation of O2 from hemoglobin is enhanced

How does pH affect T vs R

low pH stabilizes T state cause oxygen saturation drop in tissue

Hemoglobin with bound CO2 and H+ ions

carried back to lungs where they are released and oxygen rebinds

Interaction of 2,3-BPG hemoglobin

found in red blood cells, traps hemoglobin in T state, acts as a hetertropic negative effector because binding of 2,3-BPG to a secondary site inhibits the binding of oxygen to the primary site.

What does 2,3-BPG react with

polar positive a.a and both B subunit.

Where does 2,3 BPG bind

the central cavity of T state,

What happesn to this cavity when hemoglobin binds O2 and shifts to R state

2,3 BPG gets kicked out

How is O2 and 2,3 BPG in the lungs

high O2, O2 binding favors the R state, has a smaller central cavity so 2,3 BPG cant’ bind effectively

How is O2 and 2,3 BPG in the tissues

hemoglobin releases O2 and shifts to the T state, opening central cavity, 2,3 BPG binds stabilizing the T state and promoting further O2 releases

O2 and 2,3 BPG in return to lungs

high O2 concentration drives O2 binding, shifting hemoglobin back from T to R state, the central cavity narrows and 2,3 BPG is kicked out

Fetal hemoglobin

reduced 2,3 BPG binding, higher O2, acuqires O2 from maternal hemoglobin, contain y subunits instead of b, the y subunits bind 2,3 BPG less strongly, so HbF has high affinity for O2, this higher O2 affinity allows fetal hemoglobin to pull O2 from maternal hemoglobin across placenta.