Transcript Enzyme Mechanisms - Illinois Institute of Technology

Enzyme Regulation:
Globin examples
Andy Howard
Introductory Biochemistry, Fall 2008
Tuesday 4 November 2008
Biochemistry: Regulation
1
11/04/2008
Hemoglobin as an
honorary enzyme
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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.
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Mechanism Topics
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Regulation,
concluded
Allostery: more
details
Post-translational
modification
Protein-protein
interactions
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Globins as
Examples
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Biochemistry: Regulation
Oxygen binding
Tertiary structure
Quarternary
structure
R and T states
Allostery
Bohr effect
BPG as an effector
Sickle-cell anemia
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Regulation of enzymes
(review)
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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:
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Thermodynamics
Enzyme availability
Allostery
Post-translational modification
Protein-protein interactions
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Kinetics of
allosteric enzymes
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Generally these don’t obey MichaelisMenten 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
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T  R State transitions
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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
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MWC model
for allostery
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Emphasizes
symmetry and
symmetry-breaking
in seeing how
subunit interactions
give rise to
allostery
Can only explain
positive
cooperativity
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Koshland (KNF) model
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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
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Heterotropic effectors
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Post-translational modification
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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
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Zymogens
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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
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Blood clotting
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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:
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Intrinsic: blood sees injury directly
Extrinsic: injured tissues release factors that
stimulate process
Come together at factor X
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Cascade
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Protein-protein interactions
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One major change in biochemistry in the last 20
years is the increasing emphasis on proteinprotein 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
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Globins as aids to
understanding
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Myoglobin and hemoglobin are wellunderstood non-enzymatic proteins
whose properties help us understand
enzyme regulation
Hemoglobin is described as an “honorary
enzyme” in that it “catalyzes” the reaction
O2(lung)  O2 (peripheral tissues)
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Setting the stage for this story
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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 oxygenrequiring processes, particularly oxidative
phosphorylation in mitochondria in rapidly
metabolizing tissues
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Why is myoglobin needed?
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Free heme will bind O2 nicely;
why not just rely on that?
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Protein has 3 functions:
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Immobilizes the heme group
Discourages oxidation of Fe2+ to Fe3+
Provides a pocket that oxygen can fit into
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Setting the stage II
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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
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Structure determinations
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Myoglobin & hemoglobin were the first
two proteins to have their 3-D structures
determined experimentally
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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!)
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Myoglobin structure
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QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Almost entirely -helical
Sperm whale
8 helices, 7-26 residues each
myoglobin; 1.4 Å
Bends between helices generally short 18 kDa monomer
PDB 2JHO
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 sidechain N (his F8): fig.15.25
Sixth coordination site is occupied by O2, H2O,
CO, or whatever else fits into the ligand site
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O2 binding alters myoglobin
structure a little
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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!
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Hemoglobin
structure
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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
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QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Human
deoxyHb
PDB 2HHB
1.74Å
65kDa
heterotetramer
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Subunit
interfaces in Hb
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Subunit interfaces are
where many of the
allosteric interactions
occur
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Strong interactions:
1 with 1 and 2,
1 with 1 and 2
Weaker interactions:
1 with 2, 1 with 2
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Biochemistry: Regulation
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Image courtesy
Pittsburgh
Supercomputing
Center
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Subunit dynamics
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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
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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
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Conformational states
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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
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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
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Hemoglobin allostery
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Known since early 1900’s that
hemoglobin displayed sigmoidal oxygenbinding 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
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R  T states and hemoglobin
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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
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Hill coefficients
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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 O2binding sites are filled, and n, a unitless value
characterizing the cooperativity
n is called the Hill coefficient.
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PO2 and fraction oxygenated
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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 halfoccupied hemoglobin
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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!
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Real Hill parameters (p.496)
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Human hemoglobin has n ~ 2.8, P50 ~ 26 Torr
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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!
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MWC theory
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Monod, Wyman, Changeux developed
mathematical model describing TR
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 TR equilibrium,
so that tends to make more R
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Koshland’s contribution
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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
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Added complication I: pH
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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)
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How the Bohr
effect happens
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R form has an effective pKa that
is lower than T
One reason:
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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.
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Biochemistry: Regulation
Cartoon
courtesy
John
Robertus,
UT Austin
p. 34 of 44
Physiological result
of Bohr effect
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Actively metabolizing tissues tend to
produce lower pH
That promotes O2 release where it’s
needed
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CO2 also promotes
dissociation
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High [CO2] lowers pH because it
dissolves with the help of the enzyme
carbonic anhydrase and dissociates:
H2O + CO2  H2CO3  H+ + HCO3Bicarbonate transported back to lungs
When Hb gets re-oxygenated,
bicarbonate gets converted back to
gaseous CO2 and exhaled
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Role of carbamate
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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
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Another allosteric
effector
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Quic kTi me™ a nd a
TIFF (Un co mp res se d) d ec ompre ss or
ar e n ee ded to see th is p ictu re .
BPG (Wikimedia)
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!
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BPG and physiology
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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
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Fetal hemoglobin
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Higher oxygen affinity because the type
of hemoglobin found there has a lower
affinity for BPG
Fetal Hb is 22;
 doesn’t bind BPG as much as .
That helps ensure that plenty of O2 gets
from mother to fetus across the placenta
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Sickle-cell anemia
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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
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Why has this
mutation survived?
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QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Homozygotes don’t generally
Deoxy HbS
survive to produce progeny;
2.05 Å
but heterozygotes do
PDB 2HBS
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
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How is sickling related
to malaria?
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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)
11/04/2008
Biochemistry: Regulation
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Plasmodium
falciparum
from A.Dove
(2001) Nature
Medicine
7:389
p. 43 of 44
Other hemoglobin mutants
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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
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