11/04/2008 Biochemistry: Regulation 1 Enzyme Regulation: Globin examples Andy Howard Introductory...

11/04/2008Biochemistry: Regulation 1

Enzyme Regulation:Globin examples

Andy HowardIntroductory Biochemistry, Fall 2008

Tuesday 4 November 2008

11/04/2008 Biochemistry: Regulation p. 2 of 44

Hemoglobin as an honorary enzyme

We’ll illustrate some of our understandings of regulation and allostery via hemoglobin and myoglobin. But first we need to finish establishing general principles about enzyme regulation.


Mechanism Topics Globins as

Examples Oxygen binding Tertiary structure Quarternary

structure R and T states Allostery Bohr effect BPG as an effector Sickle-cell anemia

Regulation, concluded

Allostery: more details

Post-translational modification

Protein-protein interactions


Regulation of enzymes (review)

The very catalytic proficiency for which enzymes have evolved means that their activity must not be allowed to run amok

Activity is regulated in many ways: Thermodynamics Enzyme availability Allostery Post-translational modification Protein-protein interactions


Kinetics of allosteric enzymes Generally these don’t obey Michaelis-

Menten kinetics Homotropic positive effectors produce

sigmoidal (S-shaped) kinetics curves rather than hyperbolae

This reflects the fact that the binding of the first substrate accelerates binding of second and later ones


T R State transitions Many allosteric effectors influence the

equilibrium between two conformations One is typically more rigid and inactive,

the other is more flexible and active The rigid one is typically called the “tight”

or “T” state; the flexible one is called the “relaxed” or “R” state

Allosteric effectors shift the equilibrium toward R or toward T


MWC model for allostery Emphasizes

symmetry and symmetry-breaking in seeing how subunit interactions give rise to allostery

Can only explain positive cooperativity


Koshland (KNF) model Emphasizes conformational changes from

one state to another, induced by binding of effector

Ligand binding and conformational transitions are distinct steps

… so this is a sequential model for allosteric transitions

Allows for negative cooperativity as well as positive cooperativity


Heterotropic effectors


Post-translational modification We’ve already looked at phosphorylation Proteolytic cleavage of the enzyme to

activate it is another common PTM mode Some proteases cleave themselves

(auto-catalysis); in other cases there’s an external protease involved

Blood-clotting cascade involves a series of catalytic activations


Zymogens

As mentioned earlier, this is a term for an inactive form of a protein produced at the ribosome

Proteolytic post-translational processing required for the zymogen to be converted to its active form

Cleavage may happen intracellularly, during secretion, or extracellularly


Blood clotting

Seven serine proteases in cascade Final one (thrombin) converts fibrinogen

to fibrin, which can aggregate to form an insoluble mat to prevent leakage

Two different pathways: Intrinsic: blood sees injury directly Extrinsic: injured tissues release factors that

stimulate process Come together at factor X


Cascade


Protein-protein interactions One major change in biochemistry in the last 20

years is the increasing emphasis on protein-protein interactions in understanding biological activities

Many proteins depend on exogenous partners for modulating their activity up or down

Example: cholera toxin’s enzymatic component depends on interaction with human protein ARF6


Globins as aids to understanding Myoglobin and hemoglobin are well-

understood non-enzymatic proteins whose properties help us understand enzyme regulation

Hemoglobin is described as an “honorary enzyme” in that it “catalyzes” the reactionO2(lung) O2 (peripheral tissues)


Setting the stage for this story Myoglobin is a 16kDa monomeric O2-storage

protein found in peripheral tissues Has Fe-containing prosthetic group called

heme; iron must be in Fe2+ state to bind O2

It yields up dioxygen to various oxygen-requiring processes, particularly oxidative phosphorylation in mitochondria in rapidly metabolizing tissues


Why is myoglobin needed? Free heme will bind O2 nicely;

why not just rely on that? Protein has 3 functions:

Immobilizes the heme group Discourages oxidation of Fe2+ to Fe3+

Provides a pocket that oxygen can fit into


Setting the stage II Hemoglobin (in vertebrates, at least) is a

tetrameric, 64 kDa transport protein that carries oxygen from the lungs to peripheral tissues

It also transports acidic CO2 the opposite direction

Its allosteric properties are what we’ll discuss


Structure determinations Myoglobin & hemoglobin were the first

two proteins to have their 3-D structures determined experimentally Myoglobin: Kendrew, 1958 Hemoglobin: Perutz, 1958 Most of the experimental tools that

crystallographers rely on were developed for these structure determinations

Nobel prizes for both, 1965 (small T!)


Myoglobin structure

Almost entirely -helical 8 helices, 7-26 residues each Bends between helices generally short Heme (ferroprotoporphyrin IX) tightly but

noncovalently bound in cleft between helices E&F Hexacoordinate iron is coordinated by 4 N atoms

in protoporphyrin system and by a histidine side-chain N (his F8): fig.15.25

Sixth coordination site is occupied by O2, H2O, CO, or whatever else fits into the ligand site

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Sperm whale myoglobin; 1.4 Å18 kDa monomer

PDB 2JHO


O2 binding alters myoglobin structure a little

Deoxymyoglobin: Fe2+ is 0.55Å out of the heme plane, toward his F8, away from O2 binding site

Oxymyoglobin: moves toward heme plane—now only 0.26Å away (fig.15.26)

This difference doesn’t matter much here, but it’ll matter a lot more in hemoglobin!


Hemoglobin structure

Four subunits, each closely resembling myoglobin in structure (less closely in sequence);H helix is shorter than in Mb

2 alpha chains,2 beta chains



Human deoxyHbPDB 2HHB1.74Å65kDa hetero-tetramer


Subunit interfaces in Hb

Subunit interfaces are where many of the allosteric interactions occur Strong interactions:

1 with 1 and 2,1 with 1 and 2

Weaker interactions:1 with 2, 1 with 2



Image courtesyPittsburghSupercomputingCenter


Subunit dynamics 1-1 and 2-2 interfaces are solid and

don’t change much upon O2 binding 1-2 and 2-1 change much more:

the subunits slide past one another by 15º Maximum movement of any one atom ~ 6Å Residues involved in sliding contacts are in

helices C, G, H, and the G-H corner This can be connected to the oxygen

binding and the movement of the iron atom toward the heme plane


Conformational states We can describe this shift as a transition from

one conformational state to another The stablest form for deoxyHb is described as a

“tense” or T state Heme environment of beta chains is almost

inaccessible because of steric hindrance That makes O2 binding difficult to achieve

The stablest form for oxyHB is described as a “relaxed” or R state

Accessibility of beta chains substantially enhanced


Hemoglobin allostery Known since early 1900’s that

hemoglobin displayed sigmoidal oxygen-binding kinetics

Understood now to be a function of higher affinity in 2nd, 3rd, 4th chains for oxygen than was found in first chain

This is classic homotropic allostery even though this isn’t really an enzyme


R T states and hemoglobin We visualize each Hb monomer as

existing in either T (tight) or R (relaxed) states; T binds oxygen reluctantly, R binds it enthusiastically

DeoxyHb is stablest in T state Binding of first Hb stabilizes R state in

the other subunits, so their affinity is higher


Hill coefficients Recognition that binding could be modeled by a

polynomial fit to pO2

Kinetics worked out in 1910’s: didn’t require protein purification, just careful in vitro measurements of blood extracts

Actual equation is on next page Relevant parameters to determine are P50, the

oxygen partial pressure at which half the O2-binding sites are filled, and n, a unitless value characterizing the cooperativity

n is called the Hill coefficient.


PO2 and fraction oxygenated If Y is fraction of globin that is oxygenated and

pO2 is the partial pressure of oxygen,then Y/(1-Y) = (pO2 /P50)n

P50 is a parameter corresponding to half-occupied hemoglobin work out the algebra: When pO2 = P50, Y/(1-Y) = 1n=1 so Y = 1/2.

Note that the equation on p.496 is wrong!


Real Hill parameters (p.496) Human hemoglobin has n ~ 2.8, P50 ~ 26 Torr

Perfect cooperativity, tetrameric protein: n =4 No cooperativity at all would be n = 1.

Lung pO2 ~ 100 Torr;peripheral tissue 10-40 Torr

So lung has Y~0.98, periphery has Y~0.06! That’s a big enough difference to be functional If n=1, Ylung=0.79, Ytissue=0.28; not nearly as good

a delivery vehicle!


MWC theory Monod, Wyman, Changeux developed

mathematical model describing TR transitions and applied it to Hb

Accounts reasonably well for sigmoidal kinetics and Hill coefficient values

Key assumption: ligand binds only to R state, so when it binds,it depletes R in the TR equilibrium,so that tends to make more R


Koshland’s contribution

Conformational changes between the two states are also clearly relevant to the discussion

His papers from the 1970’s articulating the algebra of hemoglobin-binding kinetics are amazingly intricate


Added complication I: pH Oxygen affinity is pH dependent That’s typical of proteins, especially those in

which histidine is involved in the activity (remember it readily undergoes protonation and deprotonation near neutral pH)

Bohr effect (also discovered in early 1900’s): lower affinity at low pH (fig. 15.33)


How the Bohr effect happens

R form has an effective pKa that is lower than T

One reason: In the T state, his146 is close to

asp 94. That allows the histidine pKa to be higher

In R state, his146 is farther from asp 94 so its pKa is lower.

Cartoon courtesy John Robertus, UT Austin


Physiological result of Bohr effect

Actively metabolizing tissues tend to produce lower pH

That promotes O2 release where it’s needed


CO2 also promotes dissociation High [CO2] lowers pH because it

dissolves with the help of the enzyme carbonic anhydrase and dissociates:H2O + CO2 H2CO3 H+ + HCO3

-

Bicarbonate transported back to lungs When Hb gets re-oxygenated,

bicarbonate gets converted back to gaseous CO2 and exhaled


Role of carbamate

Free amine groups in Hb react reversibly with CO2 to form R—NH—COO- + H+

The negative charge on the amino terminus allows it to salt-bridge to Arg 141

This stabilizes the T (deoxy) state


Another allosteric effector 2,3-bisphosphoglycerate is a heterotropic

allosteric effector of oxygen binding Fairly prevalent in erythrocytes (4.5 mM);

roughly equal to [Hb] Hb tetramer has one BPG binding site BPG effectively crosslinks the 2 chains It only fits in T (deoxy) form!

QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.

BPG (Wikimedia)


BPG and physiology

pO2 is too high (40 Torr) for efficient release of O2 in many cells in absence of BPG

With BPG around, T-state is stabilized enough to facilitate O2 release

Big animals (e.g. sheep) have lower O2 affinity but their Hb is less influenced by BPG


Fetal hemoglobin

Higher oxygen affinity because the type of hemoglobin found there has a lower affinity for BPG

Fetal Hb is 22; doesn’t bind BPG as much as .

That helps ensure that plenty of O2 gets from mother to fetus across the placenta


Sickle-cell anemia Genetic disorder: Hb residue 6 mutated from

glu to val. This variant is called HbS. Results in intermolecular interaction between

neighboring Hb tetramers that can cause chainlike polymerization

Polymerized hemoglobin will partially fall out of solution and tug on the erythrocyte structure, resulting in misshapen (sickle-shaped) cells

Oxygen affinity is lower because of insolubility


Why has this mutation survived?

Homozygotes don’t generallysurvive to produce progeny;but heterozygotes do

Heterozygotes do have modestly reduced oxygen-carrying capacity in their blood because some erythrocytes are sickled

BUT heterozygotes are somewhat resistant to malaria, so the gene survives in tropical places where malaria is a severe problem



Deoxy HbS2.05 Å

PDB 2HBS


How is sickling related to malaria? Malaria parasite (Plasmondium spp.)

infects erythrocytes They’re unable to infect sickled cells So a partially affected cell might

survive the infection better than a non-sickled cell

Still some argument about all of this Note that most tropical environments

have plenty of oxygen around (not a lot of malaria at 2000 meters elevation)



Plasmodium falciparumfrom A.Dove (2001) Nature Medicine 7:389


Other hemoglobin mutants Because it’s easy to get human blood,

dozens of hemoglobin mutants have been characterized

Many are asymptomatic Some have moderate to severe effects

on oxygen carrying capacity or erythrocyte physiology

11/04/2008 Biochemistry: Regulation 1 Enzyme Regulation: Globin examples Andy Howard Introductory...

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Transcript of 11/04/2008 Biochemistry: Regulation 1 Enzyme Regulation: Globin examples Andy Howard Introductory...