Transcript Enzyme Mechanisms - Illinois Institute of Technology

Regulation II;
Molecular Motors I
Andy Howard
Introductory Biochemistry
9 November 2010
Enz Regulation II; Molecular Motors I
11/09/2010
Hemoglobin exemplifies
allostery
Even though it isn’t really an
enzyme, hemoglobin can teach us
how allostery in enzymes works.
 After that we’ll talk about
molecular motors.

11/09/2010 Enz Regulation II; Molecular Motors I
P. 2 of 67
Globins & Motor Topics

Globins as
Examples








Oxygen binding
Tertiary structure
Quarternary
structure
R and T states
Allostery
Bohr effect
BPG as an effector
Sickle-cell anemia

Molecular Motors:

Definition
Microtubules and
their partners







Tubulin
Structure
Cilia & flagella
Movement of
organelles
Dyneins & kinesins
DNA helicases
Bacterial flagella
11/09/2010 Enz Regulation II; Molecular Motors I
P. 3 of 67
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 oxygenrequiring processes, particularly oxidative
phosphorylation in mitochondria in rapidly
metabolizing tissues
11/09/2010 Enz Regulation II; Molecular Motors I
P. 4 of 67
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
11/09/2010 Enz Regulation II; Molecular Motors I
P. 5 of 67

Structure
determinations
Myoglobin & hemoglobin were the first 2
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!)
11/09/2010 Enz Regulation II; Molecular Motors I
QuickTi me™ and a
decompressor
are needed to see thi s pi cture.
Photo courtesy
EMBL
QuickTime™ and a
decompressor
are needed to see this picture.
Photo courtesy
Oregon State
Library
P. 6 of 67
Myoglobin structure






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
11/09/2010 Enz Regulation II; Molecular Motors I
P. 7 of 67
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!
11/09/2010 Enz Regulation II; Molecular Motors I
P. 8 of 67
Hemoglobin
structure


QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
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
11/09/2010 Enz Regulation II; Molecular Motors I
Human
deoxyHb
PDB 2HHB
1.74Å
65kDa
heterotetramer
P. 9 of 67
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
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Image courtesy
Pittsburgh
Supercomputing
Center
11/09/2010 Enz Regulation II; Molecular Motors I
P. 10 of 67
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
11/09/2010 Enz Regulation II; Molecular Motors I
P. 11 of 67
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
11/09/2010 Enz Regulation II; Molecular Motors I
P. 12 of 67
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
11/09/2010 Enz Regulation II; Molecular Motors I
P. 13 of 67
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
11/09/2010 Enz Regulation II; Molecular Motors I
P. 14 of 67
Binding and pO2


Hill found that 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
11/09/2010 Enz Regulation II; Molecular Motors I
Quic kTime™ and a
decompres sor
are needed to s ee t his pict ure.
Sir Archibald V. Hill
photo courtesy
nobelprize.org
P. 15 of 67
Hill coefficients



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.
11/09/2010 Enz Regulation II; Molecular Motors I
P. 16 of 67
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
4th-edition formulation: P50n  K so
Y/(1-Y) = pO2n / K
P50 is a parameter corresponding to halfoccupied hemoglobin



work out the algebra:
When pO2 = P50, Y/(1-Y) = 1n=1 so Y = 1/2.
Note that the equation on p.496 of the enhanced
3rd edition is wrong!
11/09/2010 Enz Regulation II; Molecular Motors I
P. 17 of 67
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!
11/09/2010 Enz Regulation II; Molecular Motors I
P. 18 of 67
MWC theory



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
11/09/2010 Enz Regulation II; Molecular Motors I
QuickTime™ and a
decompressor
are needed to see this picture.
Jacques Monod
Photo Courtesy
Nobelprize.org
P. 19 of 67
Koshland’s
contribution


QuickTime™ and a
decompressor
are needed to see this picture.
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
11/09/2010 Enz Regulation II; Molecular Motors I
Dan Koshland
Photo Courtesy
U. of California
P. 20 of 67
Added
complication I: pH



QuickTime™ and a
decompressor
are needed to see this picture.
Oxygen affinity is pH dependent
That’s typical of proteins, especially
those in which histidine is involved in
the activity (remember it readily
Christian Bohr
undergoes protonation and
photo courtesy
deprotonation near neutral pH)
Wikipedia
Bohr effect (also discovered in early
1900’s): lower affinity at low pH (fig.
15.33)
11/09/2010 Enz Regulation II; Molecular Motors I
P. 21 of 67
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.
11/09/2010 Enz Regulation II; Molecular Motors I
Cartoon
courtesy
Jon
Robertus,
UT Austin
P. 22 of 67
Physiological result
of Bohr effect


Actively metabolizing tissues tend to
produce lower pH
That promotes O2 release where it’s
needed
11/09/2010 Enz Regulation II; Molecular Motors I
P. 23 of 67
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+ + HCO3Bicarbonate transported back to lungs
When Hb gets re-oxygenated,
bicarbonate gets converted back to
gaseous CO2 and exhaled
11/09/2010 Enz Regulation II; Molecular Motors I
P. 24 of 67
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
11/09/2010 Enz Regulation II; Molecular Motors I
P. 25 of 67
Another allosteric
effector





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!
11/09/2010 Enz Regulation II; Molecular Motors I
P. 26 of 67
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
11/09/2010 Enz Regulation II; Molecular Motors I
P. 27 of 67
Fetal hemoglobin



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
11/09/2010 Enz Regulation II; Molecular Motors I
P. 28 of 67
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
11/09/2010 Enz Regulation II; Molecular Motors I
P. 29 of 67
Sickling and polymerization
11/09/2010 Enz Regulation II; Molecular Motors I
P. 30 of 67
Why has this
mutation survived?



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
11/09/2010 Enz Regulation II; Molecular Motors I
P. 31 of 67
How is sickling related
to malaria?





QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
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 nonsickled 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)
11/09/2010 Enz Regulation II; Molecular Motors I
Plasmodium
falciparum
from A.Dove
(2001) Nature
Medicine
7:389
P. 32 of 67
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
11/09/2010 Enz Regulation II; Molecular Motors I
P. 33 of 67
What is a molecular motor?
A protein-based system that
interconverts chemical energy and
mechanical work
 We’ll discuss several molecular
motors today, and then next Monday
we’ll look (perhaps) at the most
important one: the vertebrate
muscle.

11/09/2010 Enz Regulation II; Molecular Motors I
P. 34 of 67
Microtubules

30-nm structures composed of repeating
units of a heterodimeric protein, tubulin



-tubulin: 55 kDa
-tubulin: 55 kDa also
Structure of microtubule itself: polymer in
which the heterodimers wrap around in a
staggered way to produce a tube
11/09/2010 Enz Regulation II; Molecular Motors I
P. 35 of 67
Tubulin structure





 and  are similar but not identical
Structure determined by electron
diffraction, not X-ray diffraction
Some NMR structures available too
Two GTP binding sites per monomer
Heterodimer is stable if Ca2+ present
11/09/2010 Enz Regulation II; Molecular Motors I
P. 36 of 67
iClicker quiz question 1

Why might you expect crystallization of
tubulin to be difficult?





(a) It is too big to crystallize
(b) It is too small to crystallize
(c) Proteins that naturally form complex but
non-crystalline 3-D structures are resistant to
crystallization
(d) It is membrane-bound
(e) none of the above
11/09/2010 Enz Regulation II; Molecular Motors I
P. 37 of 67
Tubulin
dimer

G&G Fig. 16.2
11/09/2010 Enz Regulation II; Molecular Motors I
P. 38 of 67
Microtubule
structure



Polar structure
composed of / dimers
Dimers wrap around tube
as they move
Asymmetric: growth at
plus end
11/09/2010 Enz Regulation II; Molecular Motors I
P. 39 of 67
Treadmilling


Dimers added at plus
end while others
removed at minus end
(GTP-dependent): that
effectively moves the
microtubule
Fig. 16.3 / 16.13
11/09/2010 Enz Regulation II; Molecular Motors I
P. 40 of 67
Role in cytoskeleton



Microtubules have a role apart from their role
in molecular motor operations:
They are responsible for much of the rigidity
of the cytoskeleton
Cytoskeleton contains:



Microtubules (made from tubulin)
Intermediate fibers (7-12nm; made from keratins
and other proteins)
Microfilaments (8nm diameter: made from actin)
11/09/2010 Enz Regulation II; Molecular Motors I
P. 41 of 67
Cytoskeletal
components

Fig. 16.4 /16.11
11/09/2010 Enz Regulation II; Molecular Motors I
P. 42 of 67
Cilia and flagella


Both are microtubule-based structures
used in movement
Cilia:



short, hairlike projections, found on many
animal and lower-plant cells
beating motion moves cells or helps move
extracellular fluid over surface
Flagella


Longer, found singly or a few at a time
Propel cells through fluids
11/09/2010 Enz Regulation II; Molecular Motors I
P. 43 of 67
Axonemes

Bundle of microtubule fibers:





Two central microtubules
Nine pairs of joined microtubules
Often described as a 9+2 arrangement
Surrounded by plasma membrane that
connects to the cell’s PM
If we remove the PM and add a lot of salt,
the axoneme will release a protein called
dynein
11/09/2010 Enz Regulation II; Molecular Motors I
P. 44 of 67
Axoneme
structure



Inner pair
connected by
bridge
Outer nine pairs
connected to
each other and
to inner pair
Fig. 16.5
11/09/2010 Enz Regulation II; Molecular Motors I
P. 45 of 67
How cilia move




Each outer pair contains a
smaller, static A tubule and
a larger, dynamic B tubule
Dynein walks along B tubule
while remaining attached to
A tubule of a different pair
Crosslinks mean the axoneme bends
Dynein is a complex protein assembly:


ATPase activity in 2-3 dynein heavy chains
Smaller proteins attach at A-tubule end
11/09/2010 Enz Regulation II; Molecular Motors I
P. 46 of 67
Dynein movement

Fig. 16.6
11/09/2010 Enz Regulation II; Molecular Motors I
P. 47 of 67
Inhibitors of
microtubule
polymerization


Vinblastine & vincristine
are inhibitors: show
antitumor activity by
shutting down cell
division
Colchicine inhibits
microtubule
polymerization: relieves
gout, probably by slowing
movement of white cells
11/09/2010 Enz Regulation II; Molecular Motors I
P. 48 of 67
Paclitaxel: a
stimulator




Formerly called taxol
Stimulates microtubule
polymerization
Antitumor activity
Stimulates search for
other microtubule
polymerization stimulants
11/09/2010 Enz Regulation II; Molecular Motors I
P. 49 of 67
iClicker question 2
2. How do you imagine paclitaxel might
work?




(a) by producing frantic cell division
(b) by interfering with microtubule disassembly,
preventing cell division
(c ) by causing changes in tertiary structures of
 and  tubulin
(d) none of the above
11/09/2010 Enz Regulation II; Molecular Motors I
P. 50 of 67
Movement of organelles
and vacuoles




Can be fast:
2-5 µm s-1
Hard to study
1985: Kinesin
isolated
1987: Cytosolic
dynein found
11/09/2010 Enz Regulation II; Molecular Motors I
P. 51 of 67
Cytosolic dynein



Mostly moves organelles & vesicles
from (+) to (-), so it moves things
toward the center of the cell
Heavy chain ~ 400kDa, plus smaller
peptides (53-74 kDa)
Microtubule-activated ATPase activity
11/09/2010 Enz Regulation II; Molecular Motors I
P. 52 of 67
Kinesin





Mostly moves organelles from (-) to (+)
That has the effect of moving things outward
360 kDa: 110 kDa heavy chains, also 65-70 kDa
subunits (2 + 2?)
Head domain of heavy chain (38 kDa) binds ATP
and microtubule: cooperative interactions
between pairs of head domains in kinesin,
causing conformational changes in a single
tubulin subunit
8 nm movements along long axis of microtubule
11/09/2010 Enz Regulation II; Molecular Motors I
P. 53 of 67