Lecture 9 Ch 7 and 9

Allosteric Proteins and Hemoglobin

An Extremely Potent Inhibitor of

Xanthine Oxidoreductase
CRYSTAL STRUCTURE OF
THE ENZYME-INHIBITOR
COMPLEX AND MECHANISM
OF INHIBITION
Okamoto K et al. J. Biol. Chem. 2003;278:1848-1855

TEI-6720 (2-(3-cyano-4-
isobutoxyphenyl)-4-methyl-5-
thiazolecarboxylic acid) is an extremely
potent inhibitor of xanthine
oxidoreductase. Steady state kinetics
measurements exhibit mixed type
inhibition with K i and K i values of 1.2
0.05 1010 m and 9 0.05
1010 m, respectively.

Additional Factors
that Affect Enzyme Activity
1. Enzyme Expression Levels
2. Covalent Modifications
3. Environmental Factors
4. Inhibitors
5. Regulatory Molecules (Allosteric Regulators)

Certain Enzymes are Sensitive Regulatory

Points in Metabolic Pathways

Allosteric enzymes have other shapes

allos other stereos shape

Binding of modulator at one site affects substrate affinity

at other sites

The modulator may be

the substrate itself (cooperativity)
OR
a regulatory molecule other than the substrate
(allosteric regulation)

Allosteric Regulation
-control of a reaction pathway by a
mediator that converts an allosteric
protein from active to an inactive state
Cellular Thermostat
Ex. Feedback Inhibition
-As level of Isoleucine rises,
pathway needs to be turned down
-Isoleucine binds Enzyme 1 at
allosteric regulatory site and
converts to less active state

In the presence of
allosteric inhibitor,
enzyme has low affinity
for substrate

In the presence of
allosteric activator,
enzyme has high
affinity for substrate

Allosteric Enzymes Exhibit Cooperativity

The active versus inactive state of allosteric enzymes is

not only dependent upon allosteric regulators

R (relaxed, active) vs T (tense, inactive) forms

Substrate binding also affects active versus inactive

state

Often, binding of substrate to one subunit converts other

subunit(s) to active state

Why are allosteric enzymes

(as opposed to MM enzymes)
sensitive to allosteric regulation?

Allosteric Enzymes Exhibit Cooperativity

Allosteric regulation/cooperativity is possible with

quaternary proteins only

Example: Positive Cooperativity

Binding of substrate to one subunit increases
activity/affinity of remaining subunits (T R
conversion)

2 Models
Concerted Model
Sequential Model

Concerted Model
-All subunits either in R or T form

Sequential Model

Positive Cooperativity produces a

sigmoidal curve
Why?

Allosteric Enzymes Sense Signals

Allosteric Regulators
Although allosteric enzymes undergo substrate level
regulation (positive cooperativity), allosteric regulators
are critically important in maintaining appropriate levels
of enzymatic activity

Manifests as Feedback Inhibition (or positive

stimulation)

Substrate-induced
Positive Cooperativity

+S

Positive Allosteric
Regulation

+AR

Negative Allosteric
Regulation

-AR

-Substrate binds

-Allosteric regulator binds

-Allosteric regulator binds

-Traps the R (active) form

-Traps the R (active) form

-Traps the T (inactive)

form

-has greater affinity for

subsequent substrate
molecules (additional available
binding site)

-has greater affinity for

substrate molecules

-has less affinity for

substrate molecules

Is CTP a MM inhibitor or an allosteric regulator?

Positive or negative effects?

Is ATP a positive or negative allosteric regulator?

Michaelis-Menten Inhibition
vs. Allosteric Regulation
MM enzymes do not undergo allosteric regulation
Can be inhibited by toxins or drugs (MM inhibition)
Activity is affected by [S] and [P]
But NOT in the same way that allosteric regulators and [S] affect
allosteric proteins
In plot of [S] vs Vo -----hyperbolic curve

Allosteric enzymes have distinct physical and kinetic properties

Quaternary structure
Allosteric enzymes sense signals and respond
NOT the same as a MM enzyme being inhibited by a toxin or drug

Subunits display cooperativity (respond to signals such as [S] and

presence of allosteric regulators) and exist in two states (active or
inactive AKA relaxed or tense)
In plot of [S] vs Vo------Sigmoidal curve

The ability of allosteric proteins to sense and respond to signals is fundamental

to overall coordination of cellular processes

Learning Objectives

Compare and contrast oxygen binding in myoglobin vs hemoglobin

Discuss the molecular basis for cooperativity in oxygen binding to

hemoglobin

Explore allosteric regulators of hemoglobin

Become familiar with sickle cell anemia and CO effects on hemoglobin

Oxygen Transport
and Allosteric Regulation
Myoglobin
Need available oxygen for metabolism
Protein side-chains lack affinity for O2
Organometallic compounds such as
heme are more suitable

Solution:
Capture oxygen molecules with heme
sequestered inside the myoglobin
protein
Allows oxygen to diffuse through
muscle cells as needed

Myoglobin and Oxygen Binding

Myoglobin composed of 8 helices

A H

Heme is a
prosthetic
group

Sequesters oxygen using heme

prosthetic group
Hydrophobic pocket between
helices E and F
Protects iron of heme from
oxidation

Propionate groups exposed to

solvent

In Fe2+ state
Can form 2 additional
bonds, one on each side of
planar porphyrin ring
One position coordinated
with His side chain
Two vinyl groups buried in
hydrophobic pocket of myoglobin

Other available for oxygen

binding

Why hemoglobin (but not myoglobin)

is a good oxygen transporter

Myoglobin is a monomer (1 heme group)

Hemoglobin is a tetramer
4 heme groups
Allosteric protein (has quaternary structure)
Cooperativity
Allosteric Regulation

4 heme-bound subunits in
hemoglobin = 4 O2
molecules can be bound

Could Myoglobin Work

as a Good O2 Transporter?

Myoglobin
Oxygen-Bound Fraction

pO2 in lungs is about 13 kPa: it binds oxygen well

pO2 in tissues is about 4 kPa: it will not release it!

Muscle
s after
exercise

Lungs

Lungs on
oxygen
treatment

Hemoglobin

Found in RBCs---300 million molecules per RBC

Primary oxygen transport heme protein

Tetramer of two subunits (two identical dimers: a1b1, a2b2)

Each subunit is similar to myoglobin

Iron stabilized within heme prosthetic group

Sequestered in hydrophobic pocket
His F8 ligands Fe ion
His E7 forms hydrogen bond with O2

Strong hydrophobic
interactions stabilize
subunits

Dimers held in
association by weak ionic
and hydrogen bonds

Hemoglobin

High affinity for oxygen at high partial

pressure of O2 (pO2) and low affinity
for oxygen at low pO2
A plot of fractional saturation (oxygen
binding) and oxygen concentration
(pO2) is sigmoidal
Particularly responsive to changes in
pO2 at moderate concentration

Hemoglobin

Muscles
after
exercise

Lungs

Lungs on
oxygen
treatment

Hemoglobin

Half of
oxygen
released
All of
oxygen
released
Muscles
after
exercise

Lungs

Lungs on
oxygen
treatment

I cant let go
At the oxygen concentration found in muscle
tissues during the initial phases of exercise
1. Myoglobin will be highly saturated with
oxygen
2. Hemoglobin will be highly saturated with
oxygen

I cant let go
At the oxygen concentration found in muscle
tissues during the initial phases of exercise
1. Hemoglobin will release oxygen to nourish
cells
2. Myoglobin will release oxygen to nourish
cells

Learning Objectives

Compare and contrast oxygen binding in myoglobin vs hemoglobin

Discuss the molecular basis for cooperativity in oxygen binding to

hemoglobin

Explore allosteric regulators of hemoglobin

Become familiar with sickle cell anemia and CO effects on hemoglobin

How Can Hb Affinity

for Oxygen Change Like This?
Hemoglobin has multiple binding sites
And the binding sites interact with each other
Signal for cooperativity/allosteric regulation communicated
through ionic bonds (salt bridges) at the interface between
subunits

The fourth O2 molecule taken up by hemoglobin binds

with ~100 times greater affinity than first

In deoxyhemoglobin, Fe has only 5

ligands
Lies slightly outside heme ring
In association with His F8

When oxygenated, iron rises into ring

Pulls F helix as well
Rotates entire dimer
CONFORMATIONAL CHANGE

As oxygenation alters subunit

position, the hemoglobin
molecule changes in
quaternary structure

Take me with you!!

After oxygen binds the iron in the heme ring of
hemoglobin:
1. The iron atom becomes larger
2. His F8 anchors the iron atom outside the
porphyrin ring
3. The heme group dissociates
4. Ionic bonds in the alpha:beta dimer interface
are disrupted

R and T States of Hemoglobin

T = Tense state (deoxyhemoglobin)
O2 binding triggers a T R conformational change

R = Relaxed state, higher affinity for O2 than the T state

Conformational change from the T state to the R state
involves disrupting ion pairs between the dimer
interface
Low O2 state, ionic interaction at interface
Upon O2 binding, iron shifts position, moves associated His and
disrupts the interface
T form: salt bridge intact
R form: salt bridge disrupted

Recall: Dimers held

together by weak ionic
and hydrogen bonds
In taut T state, 8
more salt bridges
between subunits than
relaxed R form

Quaternary Hemoglobin exhibits

cooperativity with increasing oxygen
availability

The oxygen dissociation curve for

myoglobin has a hyperbolic shape that
reflects the fact that:
1. Myoglobin reversibly binds a single
molecule of oxygen
2. Myoglobins 4 subunits cooperate in
binding oxygen
3. The deoxy form of myoglobin is in the R-
state
4. Myoglobin has a lower oxygen affinity
than hemoglobin
5. Myoglobin has heme and hemoglobin
does not

Learning Objectives

Compare and contrast oxygen binding in myoglobin vs hemoglobin

Discuss the molecular basis for cooperativity in oxygen binding to

hemoglobin

Explore allosteric regulators of hemoglobin

Become familiar with sickle cell anemia and CO effects on hemoglobin

Hemoglobin and Oxygen Transport

In order to bind oxygen in the lungs and release oxygen in
tissues.
Hemoglobins affinity for oxygen is regulated by at least 4
factors
O2 levels (Cooperativity)
and Allosteric Regulators
CO2 levels
pH
2,3-BPG

Hemoglobin and CO2 Export

CO2 is produced by metabolism in tissues and must be exported
Diffuses to RBCs, forms carbonic acidbicarbonate
Also, exported in the form of a carbamate on N-termini of
hemoglobin subunits
Confers negative charge on terminal amino groups, which lie in
interface
Negative charge--favors salt bridge formation

Hb-NH2 + CO2

Hb-NH-COO- + H+

CO2 bound to hemoglobin stabilizes T form

Release O2

pH Effect on O2 Binding to Hemoglobin

Blood in lungs pH > blood in capillaries of metabolically active tissues
Hb affinity for oxygen dependent upon pH
Oxygen released at lower pHT state favored
Ionic bonds due to protonated of His residues at dimer interface

Oxygen sequestered at higher pH R state favored

Questions:
In lungs: Higher pH, High oxygenwhich form predominates?

In metabolically active tissues: Lower pH, Low oxygenWhich form

predominates?

pH Effect on O2 Binding to Hemoglobin

In tissues, oxyhemoglobin in R-state
(deprotonated side chains) accepts protons
released during metabolism (i.e. lactic acid or
carbonic acid)
R T conversion favors release of oxygen

In lungs, deoxyhemoglobin in T state releases

protons in higher pH environment (due to
exhalation of CO2)
T R conversion favors binding oxygen

-CO2 is transported to lungs on Hb and as bicarbonate

-H+ ions, which contribute to Bohr effect, are generated during
bicarbonate formation
bind His residues to favor T state-oxygen release

In the lungs, reduced CO2 concentration due to exhalation

-favors bicarbonate + H+ CO2 and H2O
-increasing pH leads to deprotonation of His residuesR state favored

BIG PICTURE:
High [CO2] and [H+] stabilize T form of Hb in tissues
Low [CO2] and [H+] lead to TR conversion in lungs
Collect oxygen where most abundant
Deliver oxygen where most needed

Regulation of O2 Binding by
2,3-Bisphosphoglycerate (2,3-BPG)
2,3-BPG decreases hemoglobins oxygen affinity
Binds deoxyhemoglobin and stabilizes T state
Levels regulated to address hypoxic situations
Fetal hemoglobin lacks 2,3-BPG binding site
-maternal fetal oxygen transfer

Which factors serve to release oxygen in

tissues:
1. Low CO2
2. Elevated [H+]
3. Low 2,3 BPG

Learning Objectives

Compare and contrast oxygen binding in myoglobin vs hemoglobin

Discuss the molecular basis for cooperativity in oxygen binding to

hemoglobin

Explore allosteric regulators of hemoglobin

Become familiar with sickle cell anemia and CO effects on hemoglobin

Genetic mutation in hemoglobin gene

-one nonconservative amino acid substitution
-aggregates under hypoxic conditions

Sickle cell anemia

Carbon Monoxide Poisoning

CO binds tightly to hemoglobin iron
Binding of CO on one heme site negatively affects oxygen
binding on remaining sites