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TEI-6720 (2-(3-cyano-4-
isobutoxyphenyl)-4-methyl-5-
thiazolecarboxylic acid) is an extremely
potent inhibitor of xanthine
oxidoreductase. Steady state kinetics
measurements exhibit mixed type
inhibition with K i and K i values of 1.2
0.05 1010 m and 9 0.05
1010 m, respectively.
Additional Factors
that Affect Enzyme Activity
1. Enzyme Expression Levels
2. Covalent Modifications
3. Environmental Factors
4. Inhibitors
5. Regulatory Molecules (Allosteric Regulators)
Allosteric Regulation
-control of a reaction pathway by a
mediator that converts an allosteric
protein from active to an inactive state
Cellular Thermostat
Ex. Feedback Inhibition
-As level of Isoleucine rises,
pathway needs to be turned down
-Isoleucine binds Enzyme 1 at
allosteric regulatory site and
converts to less active state
In the presence of
allosteric inhibitor,
enzyme has low affinity
for substrate
In the presence of
allosteric activator,
enzyme has high
affinity for substrate
2 Models
Concerted Model
Sequential Model
Concerted Model
-All subunits either in R or T form
Sequential Model
Allosteric Regulators
Although allosteric enzymes undergo substrate level
regulation (positive cooperativity), allosteric regulators
are critically important in maintaining appropriate levels
of enzymatic activity
Substrate-induced
Positive
Cooperativity
+S
Positive
Allosteric
Regulation
+AR
Negative
Allosteric
Regulation
-AR
-Substrate binds
Michaelis-Menten Inhibition
vs. Allosteric Regulation
MM
enzymes
do
not
undergo
allosteric
regulation
Can
be
inhibited
by
toxins
or
drugs
(MM
inhibition)
Activity
is
affected
by
[S]
and
[P]
But
NOT
in
the
same
way
that
allosteric
regulators
and
[S]
affect
allosteric
proteins
In
plot
of
[S]
vs Vo
-----hyperbolic
curve
Learning Objectives
Oxygen Transport
and Allosteric Regulation
Myoglobin
Need available oxygen for metabolism
Protein side-chains lack affinity for O2
Organometallic compounds such as
heme are more suitable
Solution:
Capture oxygen molecules with heme
sequestered inside the myoglobin
protein
Allows oxygen to diffuse through
muscle cells as needed
Heme is a
prosthetic
group
In Fe2+ state
Can form 2 additional
bonds, one on each side of
planar porphyrin ring
One position coordinated
with His side chain
Two vinyl groups buried in
hydrophobic pocket of myoglobin
Hemoglobin is a tetramer
4 heme groups
Allosteric protein (has quaternary structure)
Cooperativity
Allosteric Regulation
4 heme-bound subunits in
hemoglobin = 4 O2
molecules can be bound
Myoglobin
Oxygen-Bound Fraction
Muscle
s
after
exercise
Lungs
Lungs
on
oxygen
treatment
Hemoglobin
Strong hydrophobic
interactions stabilize
subunits
Dimers held in
association by weak ionic
and hydrogen bonds
Hemoglobin
Hemoglobin
Muscles
after
exercise
Lungs
Lungs on
oxygen
treatment
Hemoglobin
Half
of
oxygen
released
All
of
oxygen
released
Muscles
after
exercise
Lungs
Lungs on
oxygen
treatment
I
cant
let
go
At
the
oxygen
concentration
found
in
muscle
tissues
during
the
initial
phases
of
exercise
1. Myoglobin
will
be
highly
saturated
with
oxygen
2. Hemoglobin
will
be
highly
saturated
with
oxygen
I
cant
let
go
At
the
oxygen
concentration
found
in
muscle
tissues
during
the
initial
phases
of
exercise
1. Hemoglobin
will
release
oxygen
to
nourish
cells
2. Myoglobin
will
release
oxygen
to
nourish
cells
Learning Objectives
Learning Objectives
Hb-NH2 + CO2
Hb-NH-COO- + H+
Questions:
In lungs: Higher pH, High oxygenwhich form predominates?
BIG PICTURE:
High [CO2] and [H+] stabilize T form of Hb in tissues
Low [CO2] and [H+] lead to TR conversion in lungs
Collect oxygen where most abundant
Deliver oxygen where most needed
Regulation of O2 Binding by
2,3-Bisphosphoglycerate (2,3-BPG)
2,3-BPG decreases hemoglobins oxygen affinity
Binds deoxyhemoglobin and stabilizes T state
Levels regulated to address hypoxic situations
Fetal hemoglobin lacks 2,3-BPG binding site
-maternal fetal oxygen transfer
Learning Objectives