protein function - chapter 5

7/22/2019 Protein Function - Chapter 5

http://slidepdf.com/reader/full/protein-function-chapter-5 1/23

Myoglobin vs. HemoglobinFunction as a Result of Structure•

Allosteric proteins•

Enzymes•

Myoglobin : oxygen storage•

Carries oxygen through blood and delivers to tissues that need them-

Hemoglobin : oxygen transport and delivery•

O2 Binding Curve to Myoglobin (Mb) and Hemoglobin (Hb)

Molecular pharmacology

Torr because it is a gas-

Concentration of ligand that is binding to the protein-

X axis : partial pressure of oxygen•

There are two ways of binding-

The fraction of all the ligands that can be bound to the amount of protein○

The Y represents fraction bound-

(Mol Ligand)/(mg protein) = exact amount bound○

What we usually see is moles of ligand per mg of protein-

On the Y axis you can either have fraction bound or exact amount bound-

Y axis : O2 bound (amount of ligand bound)•

Follow how binding curve increases as ligand concentration increases•

Suggests different binding processes-

Binding curve of oxygen to myoglobin versus binding curve of oxygen to hemoglobin are radically differently•

Myoglobin Hemoglobin

Binding process Hyperbolic shape - rectangular hyperbola S shape - sigmoidal

Kd for ligand Has lower Kd than hemoglobin

Higher affinity for O2

Different curve shapes tell us binding process for oxygen to myoglobin and hemoglobin = different•

What Binding Curves Tell Us

Binding Process: the shape of the curve is representative

binding process.

•

Curve reaches limit called asymptote-

Maximal Binding: the asymptote approaches the binding

capacity of the protein.

•

Yvonne

Biochem I Tes

20

Protein Function - Chapter 5Thursday, October 20, 2011

Biochem 1 - Test 2



What is Kd?

Indicates binding is an equilibrium process-

L + P ↔ L ● P○

Kd = equilibrium dissociation binding constant-

Kd is a measure of the protein’s affinity for a ligand.•

Ligand is completely bound to protein at very low concentrations-

Small number divided by large number = small number○

If there is a high affinity - ligand protein complex = large, free ligand free protein = small numbers-

Small values of Kd indicate high-affinity (strong binding)•

It takes a lot of ligand to get it to bind go the protein in this equilibrium-

Large number divided by small number = large number○

Low affinity: free ligand free protein = large, ligand protein complex = small-

Large values of Kd indicate low-affinity (weak binding)•

Kd = (free ligand/free protein) / ligand protein complex•

O2 Binding Curve to Myoglobin

Curve reaches limit called asymptote-

Limit = Bmax (maximum amount of ligand that can

bound by the protein)

-

Experimentally you rarely reach Bmax because it i

expensive

-

Nonlinear curve fitting gives absolute value of Bm

(asymptote)

-

Maximal Binding: the asymptote approaches the binding

capacity of the protein.

•

1/2 Bmax = Kd-

Kd for ligand: is extrapolated from the curve as the

concentration of ligand that gives 50% maximal binding.

•

Do not memorize•

Most ligands called substrates bind toprotein have low affinity

•

Antibodies antigen complexes mostly

high affinity binding

•

Receptor ligand interactions (endogen

ligand like dopamine binding to recep

intermediate between high an low aff

binding

•

If you are developing a drug to interac

receptor they will all be high affinity b

or drug will never be developed

•

Hyperbolic shape indicates a noncooperative binding process. This m

that:

•

Biochem 1 - Test 2



X-Ray Crystal Structure of Myoglobin (Mb)

Monomeric protein.•

Eight alpha helices. (8 helix bundle)-

Covalently bound

Necessary for function

Prosthetic group : non protein○

Heme group (prosthetic group) covalently attached to protein by a coordinate covalent bond between N of proximal His and

heme.

-

Tertiary structure consisting of:•

Structure of the Heme Group

O2 binds to a single site on myoglobin or-

Oxygen binding to one site doesn't affect how o

binds to another site

Oxygen has multiple sites to bind to on myoglobin but

binds to each site with the same affinity

○

O2 binds to multiple sites with the same affinity.-

Hyperbolic shape indicates a noncooperative binding process. This m

that:

•

Right rectangular hyperbola-

Curve = hyperbolic shape•

FIGURE 5-3 Myoglobin. (PDB ID 1MBO) The eight α-helical segments (shown

as cylinders) are labeled A through H. Nonhelical residues in the bends that

connect them are labeled AB, CD, EF, and so forth, indicating the segments

interconnect. A few bends, including BC and DE, are abrupt and do not cont

any residues; these are not normally labeled. (The short segment visible betD and E is an artifact of the computer representation.) The heme is bound i

pocket made up largely of the E and F helices, although amino acid residues

other segments of the protein also participate.

Biochem 1 - Test 2



Basic building block of heme group = porphoryn ring system-

Each X can have different substituent group (R groups)-

Different substituents gives us different types of heme-

Figure A:•

In the middle you have Fe-

Form covalent bonds○

Nitrogens have two lone pairs of electrons - fall into empty d orbitals in iron-

Figure B:•

Space filling model of heme group-

Figure C:•

Fe has two open binding valences (top and bottom of lines)-

Bottom line is binding to N in His (proximal His)-

Figure D:•

Oxygenated blood is colored red-

Deoxygenated blood is blue-

Porphoryn = chromophore (absorbs light)•

How Does O2 Bind to Heme Group?

O2 binds to Fe in the heme group. Fe must be in the +2 oxidation state.•

Distal histidine forces ligands to bind at an angle.•

H-bonding between distal histidine and O2 stabilizes binding.•

CC bond = coordinate covalent bond•

FIGURE 5-5c Steric effects caused by ligand binding to the heme of

myoglobin. (c) Another view of the heme of myoglobin (derived from

PDB ID 1MBO), showing the arrangement of key amino acid residues

around the heme. The bound O2 is hydrogen-bonded to the distal His,

His E7 (His64), further facilitating the binding of O2.

FIGURE 5-1 Heme. The heme group is

present in myoglobin, hemoglobin, an

many other proteins, designated hem

proteins. Heme consists of a complex

ring structure, protoporphyrin IX, with

bound iron atom in its ferrous (Fe2+)

(a) Porphyrins, of which protoporphyr

only one example, consist of four pyrr

rings linked by methene bridges, with

substitutions at one or more of the podenoted X. (b, c) Two representations

heme (derived from PDB ID 1CCR). Th

atom of heme has six coordination bo

four in the plane of, and bonded to, th

porphyrin ring system, and (d) two

perpendicular to it.

Biochem 1 - Test 2



Between nitrogen and Histidine and iron-

Some bind to heme group○

One binds to O2 - last open slot is bound by O2○

Iron has 6 bonding orbitals-

Distal His forces any ligand binding to iron it to bind at an angle-

Oxygen = sp2 hybridized○

Oxygen binding at an angle = strong binding (no steric strain)-

H-bonding between nitrogen and histidine and oxygen stabilizes O2 binding-

When any ligand tries to bind Fe, it runs into steric hindrance from distal His•

Know: function of proximal His & distal His, how Fe is bound to heme, how heme is attached to protein, what hyperbolic binding cur

means, shape of binding curve = indicative of binding process, myoglobin and hemoglobin have different shapes (different binding

processes), how to get value of Kd from binding curve or table of data, determine Bmax from binding curve or table of data

•

Hydrophobic residues in the binding site protect Fe from oxidation to +3 state. Fe (+3) cannot bind O2. Mb is called metmyoglobi

Fe is +3.

FIGURE 5-5c Steric effects caused by ligand binding to the heme of myoglobin. (c) Another view of the heme of myoglobin (derived

PDB ID 1MBO), showing the arrangement of key amino acid residues around the heme. The bound O2 is hydrogen-bonded to the di

His E7 (His64), further facilitating the binding of O2.

Binding site = hydrophobic-

Val and Phe makes the binding site for oxygen to the heme group hydrophobic•

Where do most oxidants reside in us? - the blood-

Hydrophobic residues protect the binding site from entry by oxidizing agents-

Fe+2 = necessary in order for oxygen to bind to myoglobin○

Maintain iron in the iron +2 state-

Myoglobin in this instance = metmyoglobin○

If iron gets oxidized to +3 state - oxygen O2 cannot bind-

Necessary in order for myoglobin and hemoglobin to bind oxygen-

Hydrophobic environment for oxygen to bind is important•

Acts like a door to the binding site-

Distal histine = making everything bind to binding site at angle-

Valine : puts binding site in hydrophobic environment but also adds steric hindrance to binding•

O2 Binding Curve to Hemoglobin (Hb)

Biochem 1 - Test 2



Only has one binding site for oxygen○

Hyperbolic binding curve - noncooperative-

Myoglobin:•

O2 binds to multiple sites on Hb and○

Binding sites interact with one another

O2 (ligand) binding to one site alters affinity of other sites for the ligand.○

Sigmoidal shape indicates a cooperative binding process. This means that:-

Hemoglobin:•

Kd of O2 Binding to Hb

Kd can be extrapolated from curve as before.•

T = tense-

R = relaxed-

Kd is called “apparent” because it is a combination of O2 binding to the T-state of Hb (low-affinity state) and to the R-state of Hb (hi

affinity state).

•

Way that hemoglobin binds to O2 represents transition from low to high affinity states for O2-

Sigmoidal shape = combination of two hyperbolic curves (one representing low affinity for O2 and the other representing high affin

O2)

•

Reflects an overall affinity for O2 binding for hemoglobin○

Overall affinity is a blend of high and low affinity sites○

Is not the Kd of any one binding site of O2 to hemoglobin○

In cooperative binding processes - Kd is an apparent Kd-

Kd = concentration of ligand giving 50% Bmax•

FIGURE 5-12 A sigmoid (cooperative) binding

curve. A sigmoid binding curve can be viewed as a

hybrid curve reflecting a transition from a low-

affinity to a high-affinity state. Because of its

cooperative binding, as manifested by a sigmoid

binding curve, hemoglobin is more sensitive to the

small differences in O2 concentration between the

tissues and the lungs, allowing it to bind oxygen in

the lungs (where pO2 is high) and release it in the

tissues (where pO2 is low).

Biochem 1 - Test 2



X-Ray Crystal Structure of Hemoglobin

3° Structure of Hb’s Subunits Resembles Mb’s 3° Structure

Hb is an Allosteric Protein

Has multiple binding sites for ligands-

The binding of a ligand at one site affects the binding of a ligand at another site-

Effect is a change in the affinity of the protein for the ligand-

Allosteric Protein:•

If it is the ligand: homotropic modulation○

If it is another molecule: heterotropic modulation○

Allosteric modulators : can be the ligand, can be another molecule-

When O2 binds to hemoglobin, it increases the affinity of hemoglobin for O2 at the other binding sites - oxygen = pos

homotropic allosteric modulator of oxygen binding to hemoglobin

○

Positive modulator: increases affinity for ligand and shifts curve to left-

Negative modulator: decreases affinity for ligand and shifts curve to right-

Molecules that affect ligand binding are called allosteric modulators•

Quaternary - multiple subunits-

Made up of more than one protein-

Oligomeric protein.•

Two pairs of two subunit types○

α2β2 subunit structure.-

Each subunit is covalently bound to a heme group (as in Mb).-

This gives Hb four separate binding sites for O2.-

Quaternary structure consisting of:•

And then it wouldn't bind O2-

Heme group isn't on surface because then Fe could easily be oxidized to +3•

Green: Mb•

Blue: α-subunit of Hb•

If you superimpose them on each other, there is not much difference

the tertiary structure of the subunits

-

Red: β-subunit of Hb•

Difference in Kd of O2 for each heme group must be due to interactions

between Hb’s subunits.

•

Difference of O2 binding between hemoglobin and myoglobin isn't due to t

difference in the tertiary structure of the subunits

•

FIGURE 5-13 Structural

changes in a multisubun

protein undergoing

cooperative binding to

Structural stability is not

uniform throughout a p

molecule. Shown here is

Biochem 1 - Test 2



This happens throughout the body every second for your to function normally-

Waggle is between high and low affinity conformations•

Positive cooperativity-

Protein is under affect of positive allosteric modulator-

Everything runs nice and neatly-

The description in the grey boxes are describing positive allosteric modulators•

There is conformational flexibility•

There is a lot of waggle in the top picture-

When ligand binds - it locks one subunit into the conformation (interactions are altered)○

Alteration of interaction between subunits results in less waggle

Waggle is limited to various conformations of the high affinity state of the binding site (easier for ligand to bind

Once that ligand binds → binding site is locked in high affinity for the ligand□

When the second ligand binds, it is binding to a region that has higher affinity for the ligand

Locking alters interactions○

In the second picture, at the binding site - it is locked into conformation-

Ligand binds to one binding site and the binding site stabilizes the high affinity state in that binding site•

Wednesday, October 26, 2011

Hb is an Allosteric Protein

Negative allosteric modulators yield a negatively cooperative process-

Postive allosteric modulators yield a positively cooperative process-

If n < 1, binding is negatively cooperative○

If n > 1, binding is positively cooperative○

No modulation of ligand binding

If n = 1, binding is not cooperative (eg. the binding of O2 to Mb)○

Cooperativity can be quantitated by the Hill coefficient (n)-

Allosteric binding processes are cooperative•

Hemoglobin = allosteric protein•

Curve with modulator is positive - curve is shifted to the left (lower value of Kd)•

If n=1, you get the hyperbolic curve equation•

Know how to determine based off of graphs and charts•

Structural stability is not

uniform throughout a p

molecule. Shown here is

hypothetical dimeric pro

with regions of high (blu

medium (green), and low

stability. The ligand-bind

sites are composed of b

high- and low-stability

segments, so affinity for

ligand is relatively low. T

conformational changes

occur as ligand binds co

the protein from a low-

high-affinity state, a for

induced fit.

Biochem 1 - Test 2



Mathematical Descriptions of Binding Processes

Dashed line = if you calculate Kd from the graph and plug it into the hyperbolic curve it doesn't fit very well•

Hyperbolic Curve•

Describes myoglobin-

Hill Equation-

Sigmoidal Curve•

Measure of cooperatively-

n = hill coefficient•

B = bound•[L] = ligand concentration•

Subunit Interactions in HbConformational Changes Alter Affinity for O2•

Cooperatively must be due to interactions of subunits in hemoglobin•

Blue hemoglobin•

T state-

Deoxyhemoglobin•

Biochem 1 - Test 2



Look at this picture in color•

Heme group is more angled-

Porphyrin ring system = cupped-

Blue = position of helix F in T state (deoxyhemoglobin)•

Porphyrin ring system = flattens-

Has to deal with steric hindrance between porphyrin ring system and proximal histidine○

Oxygen binding to iron alters the electronic configuration of porphyrin ring system so it flattens out-

Electronic rearrangement when system flattens-

Oxygen has a lot of electron density○

Resistance for oxygen binding

Once oxygen binds the ring system goes flat and the repulsion between electron densities is removed

Electron density trying to come in at an angle to an electron dense environment → cupped porphyrin ring system ma

little difficult for oxygen to bind because electron dense + electron dense = kind of repulsive

○

Porphyrin ring system has a lot of conjugated double bonds so it is very electron dense-

When oxygen binds, porphyrin ring system undergoes electronic rearrangement (flattens), iron physically mov

mechanically yanks the helix into a different position

Proximal histidine is moved as well → proximal histidine = attached to helix → helix is pulled as well○

Iron is physically moves when porphyrin goes flat-

Red = position of helix F in R state (oxyhemoglobin)•

This picture shows conformational changes when oxygen is bound•

Oxygen bound = red representation-

When oxygen isn't bound → heme group in porphyrin ring system of heme group looks like blue representation•

Red hemoglobin•

R state-

Oxyhemoglobin•

Conformational breathability•

It contracts when it goes from T to R state (conformational change-

Central cavity in hemoglobin : when hemoglobin = in high affinity state it

contracts (center of picture)

•

-

Biochem 1 - Test 2



Low affinity-

T state = deoxyhemoglobin•

High affinity-

R state = oxyhemoglobin•

Other porphyrin ring system becomes a little bit flatter due to conformation change○

Those interactions pushes iron down a little bit and heme ring flattens out

If heme ring flattens out it removes the electron density repulsion with oxygen → a little easier for the next ox

bind with the next heme group

Iron shifting causes interaction with other subunits in hemoglobin○

Physical shift in helix-

What happens when helix shifts?•

Steric hindrance to binding site and forces all ligands to bind to Fe at an angle-

For maintenance of iron in +2 oxidation state → heme group is in hydrophobic environment○

Valine affects binding (and puts it in a hydrophobic environment)-

After second one binds, porphyrin ring goes a little bit flatter → the other two valines open a bit wider → the t

oxygen has an easier time to bind = higher affinity

The fourth one has the easiest time to bind

This is all going on right now in your body

After the first oxygen binds, the rest of the heme group goes flatter and removes the electron repulsion and the valin

other binding sites on hemoglobin swings out of the binding site a little so oxygen has an easier time coming in - the n

oxygen has an easier time binding to hemoglobin (higher affinity for hemoglobin)

○

Valine also causes steric hindrance and acts like a door to the binding site-

Valine (distal histidine) : provides steric hindrance•

Friday, October 28, 2011

Different electronic configuration in the heme ring - when oxygen is not bound to the iron in the heme ring-

Why is deoxygenate blood blue?•

Salt bridges in the box above-

In the picture above, the salt bridges stabilize the T state•

There is one between Asp and His-

In one helix there is a salt bridge within the helix•

FIGURE 5-11 Changes in conformation near heme o

binding to deoxyhemoglobin. (Derived from PDB ID

and 1BBB) The shift in the position of helix F when h

binds O2 is thought to be one of the adjustments th

triggers the T → R transition.

FIGURE 5-9a Some ion pairs that stabilize the T stat

deoxyhemoglobin. (a) Close-up view of a portion of

deoxyhemoglobin molecule in the T state (PDB ID 1H

Interactions between the ion pairs His HC3 and Asp

of the β subunit (blue) and between Lys C5 of the α

subunit (gray) and His HC3 (its α-carboxyl group) of

subunit are shown with dashed lines. (Recall that HC

the carboxyl-terminal residue of the β subunit.)

Biochem 1 - Test 2



His - Asp salt bridge is in one subunit○

His is at carboxyl terminus of helix = carboxyl terminus○

Alpha carboxyl - Lys salt bridge = between subunits

Alpha carboxyl group involved in salt bridge with Lys on another subunit○

There is a second salt bridge in the helix-

Vast majority of His = normally unprotonated○

In salt bridge carboxyl terminus is carboxyl group that is negative at 7.4

Lys : pKa = 10 (positive at 7.4)

Why could it be protonated?○

The His becomes positive because it is affected by the presence of other amino acids□

Salt bridge = ionic interaction between the two□

The environmental effect = the closet proximity of the Asp acid raises pKa of His → His becomes

protonated and salt bridge forms

There has to be some sort of environmental effect that affects the pKa of His□

How could the majority be protonated?

His pKa = 6 (95% unprotonated)○

His is positive due to environmental effects○

His = positively charged-

At 7.4 it's normal for Asp to be positively charged○

Asp = negatively charged-

Salt bridge: pH 7.4 (working tissue = pH 7.2 at the lowest)•

There are other salt bridges that are broken as well (besides the two we've learned) but they are hard to see - don't need to know t•

Good because you want R state hemoglobin in the lungs to bond oxygen → when it comes out of the lungs to t

tissues it releases oxygen

H-bonds less strong bond○

Stabilized by ionic interactions → R state is stabilized by hydrogen bonding between other residues-

Relaxed/easy going-

R state:•

Tense/rigid-

Held together by salt bridges-

T state:•

FIGURE 5-9b Some ion pairs that stab

the T state of deoxyhemoglobin. (b)

Interactions between these ion pairs,

between others not shown in (a), are

schematized in this representation of

extended polypeptide chains of

hemoglobin.

Biochem 1 - Test 2



FIGURE 5-10 The T→ R transition. (PDB ID 1HGA and 1BBB) In these depictions of deoxyhemoglobin, as in Figure 5-9, the α subunit

blue and the β subunits are gray. Positively charged side chains and chain termini involved in ion pairs are shown in blue, t heir nega

charged partners in red. The Lys C5 of each α subunit and Asp FG1 of each β subunit are visible but not labeled (compare Figu re 5-9

that the molecule is oriented slightly differently than in Figure 5-9. The transition from the T state to the R state shifts the subunit p

substantially, affecting certain ion pairs. Most noticeably, the His HC3 residues at the carboxyl termini of the β subunits, which are i

in ion pairs in the T state, rotate in the R state toward the center of the molecule, where they are no longer in ion pairs. Another dra

result of the T → R transition is a narrowing of the pocket between the β subunits.

There is a salt bridge (His-Asp - in one subunit)-

There is a carboxyl terminus salt bridge-

T state:•

You can find the same salt bridges on the bottom (other side of the molecule as T state)-

His becomes unprotonated/neutral again○

Salt bridge between carboxyl terminus and Lys → charge states don't change (because pKa dictate that they'll be posi

negative at that pH)

○

In conformational changes → His moves away from Asp in one subunit-

Hemoglobin releases oxygen○

His moves close to Asp○

Asp raises pKa of His → His becomes protonated/positively charged○

Salt bridge forms○

In the lungs → His = in R state - as hemoglobin goes out of lungs and starts going towards working tissue-

R state:•

O2 Binding Curve to Mb and Hb

Models of O2 Binding to Hb

Myoglobin and hemoglobin - different binding

processes

•

We can model oxygen binding to hemoglobin•

Biochem 1 - Test 2



Models of O2 Binding to Hb

Sequential Model proposed by Koshland•

Start with all subunits in T-state•

Binding of first O2 to first subunit alters the conformation of others to become more “R -like”•

As more O2 binds, remaining subunits resemble R-state even more until all are high-affinity•

When one molecule of oxygen binds (S = oxygen) - subunit that it binds to = converted to the R state-

Changes in subunit interactions convert joining subunits - they become more R like, partially T like-

As the second molecule of oxygen binds, all the subunits become more R like than T like-

As the third subunits bind - all of them are R state, high affinity (one being really R with a little T)-

When the last molecule of oxygen binds all of the subunits are in the R state-

Hemoglobin unbound to oxygen = purely in the T state•

The sequential model says there is a gradual transition based on subunit interactions in hemoglobin-

Both of the models are described by fairly complex mathematical binding equations-

The concerted model (previous picture) says there is no gradual transition between T and R - it is either all T or all R•

Subunits in equilibrium between T- and R-state-

Hemoglobin has conformational flexibility between T an R sta○

O2 can bind to either state of subunit (T or R)-

Proposed by Monod, Wyman, and Changeux-

Hemoglobin is free to fluctuate between T an R states whethe

oxygen is bound to it or not

○

Reflects transformation from low to high affinit

Sigmoidal binding curve = result of constant fluctuatio

Hemoglobin constantly fluctuates between T and R states○

Binding of oxygen to subunit in T state doesn't change it into the R st-

Concerted Model•

Biochem 1 - Test 2



Models are mathematically distinct but cannot be resolved experimentally.•

The truth of the binding process lies somewhere in between the two models.•

Line = calculate the variance between actual data points and the line that is drawn to the data points-

Check to see if the two models gave you about the same amount of variance○

Whichever gives the smaller variance = it is more representative○

Sum up variance and you can say that the sequential model give you X amount of variance and the concerted model gives yo

amount of variance

-

Measure binding of hemoglobin to oxygen and do nonlinear curve fitting = graph above•

Mathematically the models are equivalent•

Evolutionary Advantage to Hb ConformationSurvival in Current Environmental Conditions•

O2 Binding to Hb in the Presence of CO

O2 binding to hemoglobin the presence of carbon monoxide•

Carbon monoxide → binding orbitals in straight line-

O2 → binding orbitals at angle-

The strongest bind is the carbon monoxide coming straight (vertical) into the binding sites○

Iron → there are two binding orbitals that are at 90 degrees planar to the iron-

Carbon monoxide decreases the amount of oxygen bound - carbon monoxide had a much greater affinity for the iron in the heme gthan oxygen

•

Hemoglobin only has 50% of its binding sites occupied by carbon monoxide - it stays high affinity and doesn't really release oxygen

tissues

•

Oxygen delivery is about the same-

Mutation in red blood cells○

Decrease in oxygen binding to hemoglobin caused by:-

In anemic individuals - much less oxygen bound•

BOX 5-1 FIGURE 2 Several oxygen-binding curves: fo

normal hemoglobin, hemoglobin from an anemic

individual with only 50% of her hemoglobin function

and hemoglobin from an individual with 50% of his

hemoglobin subunits complexed with CO. The pO2 ihuman lungs and tissues is indicated.

Distal histidine forces every ligand to bind at an•

When it is bent it has about 100 t

affinity than oxygen in heme grou

○

Carbon monoxide without the distal hist

about 1000 greater affinity for iron in he

group

-

Carbon monoxide forced to bind at angle so the

strength of the bond between carbon monoxide

iron = weakened

•

O2 likes to bind at an angle and the H-bo

is stabilizing

-

CO2 is binding at an angle so it is a weaker bond•

If you increase the concentration of carbon mo

oxygen will be displaced and not rebound and y

suffocate

•

Biochem 1 - Test 2



Deficiency in red blood cells○

= insufficient hemoglobin○

Iron deficiency (oxygen needs iron to bind)○

Protected by distal histidine-

Binding site for oxygen-

Distal histidine forces oxygen to bind at an angle-

Evolutionary advantage: we don't have a heme group sitting on the surface of hemoglobin (it is in a hydrophobic binding site)•

The Bohr EffectEfficient O2 Delivery Through Allosteric Regulation•

Low [H+] = normal pH (7.4)-

Low [CO2]-

Lung:•

High [H+] = pH (7.2)-

Tissues don't have sufficient oxygen for mitochondria to utilize in production of ATP so to get sufficient energy

glycolysis, tissues produce lactic acid (carboxylic acid)

Lactic acid is released into the blood stream (pKa about 4)

Becomes unprotonated and releases H+ out

[CO2] increases because lactic acid (anaerobic conditions)○

You have an active metabolism - part of metabolism = tricarboxylic cycle

Tricarboxylic cycle processes nutrients and breaks them down in order for mitochondria to make ATP

Byproduct of tricarboxylic cycle = carbon dioxide

Because you generate bicarbonate buffer → release of a lot of lactic acid into blood stream doesn't low

by a lot

□

CO2 also binds to hemoglobin□

Carbon dioxide = released into blood stream and part of it becomes part of bicarbonate buffer system

Concentration of CO2 is high because:○

High [CO2]-

Working tissue:•

Hemoglobin has lower affinity for oxygen under conditions of low pH (high hydrogen concentrations) and high carbon dioxide

concentrations

-

Hemoglobin has high affinity for oxygen under conditions of low [H+] or high pH (normal) and under conditions of low carbon dioxid

concentration

•

As it leaves the lungs it goes to working tissues → high concentrations of H+, high CO2 → low affinity → dumps its oxygen-As hemoglobin moves out from lungs, pH of carbon dioxide concentrations makes it high affinity for oxygen → binds to oxygen•

Biochem 1 - Test 2



O2 binding to hemoglobin under various pH conditions

● •

Negative-

Because you have hydrogen ions making

pH lower and the shift to the right = neg

allosteric modulator

-

Is hydrogen ion a positive or negative allosteric

modulator and why?

•

pH 7.4 = normal blood pH (reference curve)•

pH 7.2: Lower pH = increasing [H+], curve shifte

right to higher values of kD = lower affinity(indicative of negative allosteric modulator)

•

pH 7.6 : higher pH, lower [H+], removing negati

allosteric modulator - when you remove a nega

allosteric modulator, hemoglobin has a higher

affinity for oxygen

•

Not under effect of negative modulator-

Stripped hemoglobin = sigmoidal•

Negative allosteric modulator (binding curve

shifted right)

-

Hemoglobin with CO2•

Negative allosteric modulator○

BPG = Bisphosphoglycerate-

Hb + BPG•

In blood O2 binding to hemoglobin is a positively

cooperative process under regulation of negative

allosteric modulations

•

Binding of O2 to hemoglobin reflects positive proces

process is under regulation of negative allosteric

modulators so that blood effectively delivers blood t

working tissue

•

Biochem 1 - Test 2



Monday, October 31, 2011

Adaptation to High Altitude - BPG

Highly negatively charged-

BPG = negative allosteric modulator•

People who climb mount Everest will go stay at high altitudes before hand so that their body can adjust to the decrease in ox

levels

-

Body synthesizes BPG as an adaptation for the loss of oxygen-

BPG = our adaptation to high altitudes•

More BPG in blood when there is less oxygen•

Low affinity state has large central cavity-

So BPG binds to large central cavity of the T state od hemoglobin○

BPG has a lot of negative charges on it - central cavity has a lot of positive charges-

BPG stabilizes the T state hemoglobin - low affinity state•

BPG physically inhibits the transition from T to R state and keeps hemoglobin in a low affinity state-

BPG acts like a stick being pressed into something that is open and when the thing tries to compress, it can't anymore•

The more BPG synthesized, the more hemoglobin molecules are in a low affinity state•

Low affinity state of oxygen = adaptation to decrease in oxygen in atmosphere•

Quaternary Structure of Deoxy Hb (T-state)

Image on left: large central cavity•

Image on right: BPG in center•

Binding curves of O2 to Hb

Working tissue = high concentration of negat

modulators (makes hemoglobin dump O2 int

tissue faster than it would if it were just base

equilibrium)

-

process is under regulation of negative allosteric

modulators so that blood effectively delivers blood t

working tissue

Biochem 1 - Test 2



Hemoglobin transfers over to working tissue and releases its oxygen (still kind of R state)-

Less oxygen in air means less oxygen bound to hemoglobin•

With lower oxygen binding to hemoglobin, you are delivering about 30% of the normal oxygen that should be delivered there•

At high altitudes, it only dumps 30%-

You have a shortage of 8% of oxygen delivery-

At normal oxygen, hemoglobin dumps 38% of the oxygen bound•

Hemoglobin bound to BPG will bind to less oxygen in the lungs-

You are only 1% short of oxygen delivery (this is tolerable)○

Low affinity state = more oxygen is released than normal hemoglobin → hemoglobin bound to BPG dumps 37% of its bound -

BPG: low affinity state for oxygen does not go back no matter what the concentration is•

Sickle-cell AnemiaSingle Point Mutation Leads to Serious Disease•

Supposed to be an evolutionary adaptation to promote survival in areas with malaria-

Since the protozoan that causes malaria is in red blood cells, if red blood cells are partially destroyed, protozoan can't live in -

Sickle cell anemia = genetic adaptation to improve survival in areas with high incidence of malaria•

FIGURE 5-19 A comparison of (a) uniform

cup-shaped, normal erythrocytes with (b

the variably shaped erythrocytes seen in

sickle-cell anemia, which range from nor

to spiny or sickle-shaped.

Biochem 1 - Test 2



As more an more people mate in the area and they have the gene for sickle cell anemia, you will have both par

having the gene so the children have full blown sickle cell anemia → caused more problems than malaria

That is okay if only one parent has genes for sickle cell anemia because then you only have sickle cell trait (you don't

blown anemia) - you have resistance to malaria

○

cells → you don't get malaria

Meant to be a genetic adaptation but it didn't really work out the way it was supposed to-

Single point mutation gives you sickle hemoglobin (HbS)-

Normal hemoglobin = HbA-

Negatively charged

HbA

E: glutamate○

Nonpolar

HbS

V: valine○

Single point mutation is E → V-

Single point - change in 1 amino acid in hemoglobin•

Don't have nuclei-

Have hollow shape in center-

Flexible-

Characteristics of normal red blood cells•

When blood enters capillaries → the membrane of normal RBC is willing to squish together and flex and compact/move easily throu

capillaries

•

Rupture of red blood cell → leads to anemia conditions○

Formation of clots in capillaries → becomes the real problem with sickle cell anemia (leads to tissue necrosis)○

Rigidity can lead to:-

In sickle RBC → cells are distorted/lengthened - they tend to be rigid•

What causes the sickling of red blood cells?

HbS aggregates and forms insoluble rod like structures in the RBC → causes misshapen forms of RBCs → leads to rigidity → l

clots and tissue necrosis → leads to rupturing of RBC and anemia

-

Sickle RBC is full of fibers → fibers = aggregated hemoglobin S•

Fibers don't form when hemoglobin is in R state-

When RBC comes back into lungs, it remains T state hemoglobins →misshapen RBCs○

But fibers form from T state and then they stabilize in T state → once you have the forming of the hemoglobin fiber in the T s

stays T state

-

Fibers from the T-state HbS and stabilize HbS in T state•

Biochem 1 - Test 2



Binding pocket opens up → hydrophobic-

1 amino protrusion → glutamate coming out, water interacts with it○

In normal adult hemoglobin, 1aa = glutamate-

In Hemoglobin S, 1aa that protrudes = valine-

T state surface:•

Repulsive interaction prevents binding and aggregate-

Glutamate won't bind to hydrophobic pocket because negatively charged environment = repulsive•

Once hydrophobic effect drives them together, they are stabilized by London forces → tend to stay together○

Hydrophobic effect drives two hemoglobins to bind together → you have aggregation of hemoglobin-

Valine will aggregate to hydrophobic pocket•

A lot of T state hemoglobin = sickle crisis (bad disease)-

People with sickle cell are okay if they are not too active → once they become physically active (O2 drops) and have a lot of physica

exertion - they have a lot of T state hemoglobin

•

Hydrophobic binding pocket•

Val = point mutation•

Precipitous effect in sickle cell anemia gives us the fibers•

Biochem 1 - Test 2



Strong hydrogen bonder○

Denatures proteins○

What drives the hydrophobic effect? - the gain in entropy of water when nonpolar molecules are exclud□

Why is it a chaotropic agent? - breaks up the hexagonal array of water and add entropy to water's structure

Chaotropic agent○

HbF = fetal hemoglobin → higher affinity for oxygen than HbA-

By replacing hemoglobin A with HbF□

Doesn't release oxygen as readily → prevents formation of the T state and prevents formation of the hemoglob

fibrils

-

Stimulates gene expression□

Transcriptional activator-

Acts to stimulate synthesis of HbF○

How does it work?-

Only current FDA approved treatment for sickle cell anemia•

They aggregate together and from a spiral that precipitate out o

the cell itself

-

Precipitous effect in sickle cell anemia gives us the fibers•

FIGURE 5-20b Normal and sickle-cell hemoglobin. (b) As a result o

change, deoxyhemoglobin S has a hydrophobic patch on its surface

which causes the molecules to aggregate into strands that align in

insoluble fibers.

Individual cells precipitate together•

Interaction between the molecules to give us strands•

Strands interact together and crystallize out of solution•

Biochem 1 - Test 2



Need to give in large doses for it to work•

Hydroxyurea induces cancer (as doses that are required to be used as treatment for sickle cell anemia)•

protein function - chapter 5

Documents