Transcript Document

Is ubiquitination always the
result of mistakes?
The N-end Rule: The N-terminal amino
acid determines half-life
Destabilizing N-termini
are recognized by a
special E2/E3
The eukaryotic
cell cycle is
controlled by the
ubiquitin
pathway
During the cells cycle
synthesis or mitosis
DNA damage signals cell
cycle arrest.
The Mdm2 E2/E3 keeps
p53 abundance low
under normal
conditions.
After DNA damage p53
is stabilized and it
causes the trancription
of a CDK inhibitor,
thereby stopping the cell
cycle.
Let’s examine a real world example: The globins
Oxygen Carriers
Hemerythrin
Hemocyanin
Globins
Need metals to bind to oxygen…..why?
Oxygen is a diradical
It has 2 unpaired electrons
1/23O2 + 1X ---> 1XO
This is the oxygen paradox
The spin restriction limits the
chemical reactivity by imposing a
kinetic barrier
Singlet oxygen in the excited state
is extraordinarily reactive
This is the basis for photodynamic
therapy
Metals cause oxygen to become reactive
because they are radicals themselves. They
eliminate spin restrictions
Fe(II)-O2
Fe(III)-O2-
Fe(III)-O2- + Fe(II)
Fe(III)-O22--Fe(III)
Fe(III)-O22--Fe(III)
2Fe(IV)=O Highly reactive!
2Fe(IV)=O
Fe(III)-O-Fe(III)
A picket-fence Fe(II)–porphyrin complex with bound O2-
Metals, along with proteins, can harness the reactivity of
oxygen by activating it an shielding it
Fe(II) binds dioxygen Fe(III) does not
Why?
Oxygen to metal charge transfer
Fe(II)-O2
Fe(III)-O2-
Stable
Fe(III)-O2
Fe(IV)-O2-
Unstable
Fe(II) will also bind NO, CO, S2- , CN-
The visible absorption spectra of oxygenated and
deoxygenated hemoglobins.
Distal
Proximal
C-terminus
N-terminus
Fractional saturation of myoglobin with oxygen
Hemoglobin binds oxygen cooperatively
This means that the binding of one oxygen to one
subunit affects the binding to another subunit
The two state model of hemoglobin binding
Oxy or
R state
Deoxy or
T state
Major Structural differences upon
oxidation of hemoglobin
Fe moves from 0.55Å out of the heme
plane to 0.22Å out of the plane
Extensive a1-b1 contacts unchanged
Minimal a1-b2 contact altered by as
much as 6 Å
15º offcenter rotation of the protomers
High spin Oh Fe2+
x2-y2
Low spin Oh Fe3+
x2-y2
z2
z2
xz
yz
xy
Increased radius
xz
yz
xy
Decreased radius
Ion pairs that stabilize the T-state
1) Intra-b subunit His-Asp pair
2) a Lys-b-C-terminus pair
3) Inter-a subunit Arg-Asp/C-terminus-Lys pairs
4) Inter-a subunit N-terminus-C-terminus pair
High CO2 in tissues decreases the pH: the Bohr effect
CO2 + H2O ---> H+ + HCO3-
Low pH stabilizes the T state. How?
a Lys-b-C-terminus pair
Intra-b subunit His-Asp pair
At low pH His146 is protonated allowing the ion pair to form
R-NH2 + CO2
R-NH-COO- + H+
a-amino terminus
Carbaminohemoglobin
COO-
Inter-a subunit Arg-Asp/C-terminus-Lys pairs
deoxyHb can also bind chloride ion tightly
Cl- is higher in veins than in arteries
High Cl- will cause O2 release
Inter-a subunit Arg-Asp/C-terminus-Lys pairs
Thus the T state is stabilized by:
Low pH
High CO2
High Cl-
Comparison of the O2-dissociation curves of
“stripped” Hb and whole blood in 0.01M NaCl at pH
7.0.
2,3-bisphosphoglycerate binds deoxyHb
BPG
Keeps Hb deoxygenated
-O
O
C
H
C
OPO32-
H
C
OPO32-
H
Binding of BPG to
deoxyHb.
The effect of high-altitude exposure on the p50 and
the BPG concentration of blood in sea level–
adapted individuals.
Notice: 8 mM BPG results in less saturation at high
altitude….but….results in equivalent release of O2. Note 38% release of
O2 at sea level with 5 mM BPG and 30% release at high altitude with 5
mM BPG. Also note 37% release at high altitude with 8 mM BPG!
Fetal hemoglobin (a2g2)
Neonatal hemoglobin (z2e2)
Adult hemoglobin (a2b2)
1% adult hemoglobin (a2d2)
Why are there different globins?
Myoglobin has a higher affinity for O2 in tissues
Fetal hemoglobin (a2g2)
No affinity for BPG
Thus it will look more like myoglobin