31 - Allosterism & Cooperativity Flashcards
Allosterism Definition
A change in activity & conformation of a biopolymer at a primary site
usually resulting from the binding of a compound @ a secondary site
1) >2 different kinds of sites
2) possibly 2 different kinds of binding compounds
3) interactions between 2 sites
Allosteric Effectors
LIGAND for an allosteric protein
- *Small molecules** that modulate the conformation & activity of:
- *Enzymes** / Receptors / Proteins
What is the purpose of ALLOSTERISM?
to alter the overall activity
changes at 1 site –> affect the entire assembly / DRASTIC changes
PROMOTE SENSITIVITY
to changing cellular conditions, an Evolutionary advantage
What is COOPERATIVITY?
NOT the same as Allosterism
Occurs when the
binding is NOT INDEPENDENT**, but is **INTERACTIVE
Cooperativity can arise without any shape change
but is often associated w/ allosteric effects
2 types:
Positive = augment/INCREASE binding of second molecule
Negative = reduces binding of second ligand
POSITIVE Cooperativity
when the binding of 1 ligand
AUGMENTS** or **INCREASES
the binding of the second molecule
Negative Cooperativity
when the binding of the 1st ligand
REDUCES
binding by the 2nd ligand
NOT inhibition, just reduces the binding
Hemoglobin & O2 Binding
A model for BOTH Allosteric & Cooperative System
Hb binds 4 molecules of O2 w/ POSITIVE cooperativity
binding of oxygen -> sigmoid curve
Allosterism = Conformational change in Protein
Cooperativity = Changes in O2 AFFINITY
Heme Oxygenation
Central Iron is held by 5 nitrogens
= 4 nitrogens + 1 Extra-N from HISTADINE
OXYGEN = SIXTH(6) BINDING
that tugs the nitrogen –> FLAT SHAPE = Oxygenative heme
Allosterism come BEFORE
without the oxygen group, the heme is in an ODD shape
the ODD SHAPE –> induces the BINDING of the Oxygen
POSITIVE COOPERATIVITY
T State of Hemoglobin
- Taut Conformation** = *DE-Oxygenated
- before any OXYGEN is bound*, the subunits are in a low affinity state
the shape WANTS to be bound by Oxygen, once the Oxygen binds there will be a Positive Cooperativity inducing the next Oxygen binding
R-State of Hemoglobin
Relaxed Conformation = OXYGENATED
there is a strong tendency for
neighboring HEME subunits (that DO NOT have Oxygen bound yet)
to swith from T -> R
Binding of the second O2 –> Increases the **affinity for the 3rd O2
- -> then AGAIn for the 4TH O2**
- *POSITIVE COOPERATIVITY**
BOHR EFFECT
INVERSE Relationship of CO2 & Hemoglobin’s Oxygen Affinity
more CO2 -> more basic ^pH
Protons as ligands / change in pH
HIGH Alkalinity (BASIC, CO2) = Higher Slope/Affinity
= wants to take in MORE OXYGEN
low alkalinity (acidic / low CO2) = weaker slope / lower affinity
for oxygen
MCW Model
one of 2 Models for combining Allosterism & Cooperativity
CONCERTED model
enzyme has 2 or MORE IDENTICAL SUBUNITS
subunits switch between 2 or more conformations = R & T
each subunit has >1 binding sites
ALL OR NONE
concerted manner, in the same enzyme:
ALL SUBUNITS R or ALL T
Explaination of the MCW Model
one of 2 Models for combining Allosterism & Cooperativity
CONCERTED MODEL
SIMULTANEOUS conformation change
Activators
bind to the R Form & increase its concencentration
at the expense of T-Form –> RAISE ACTIVITY / SENSITIVITY
Inhibitors
bind to the T form & make the transition to R more difficult
shift the activity curve to the RIGHT –> LESS SENSITIVITY
KNF Model
one of 2 Models for combining Allosterism & Cooperativity
SEQUENTIAL model
Subunits change one at a time
More GRADUAL vs the MWC model
more REALISTIC
Allows for a MIXTURE of conformational states of subunits
Can have mixed R/T states
often combined with Induced Fit Concept
Which model for
Allosterism & Cooperativity
Is the DIAGONAL / VERTICAL?
Diagonal = KNF
Induced Fit / Sequential Model
Vertical = MWC
All-or-none shape change
ATCase Function
Aspartate Transcarboylase = first unique step in
Pyrimidine Biosynthesis
Allosteric Enzyme that changes shape / activity in a CONCERTED FASHION (MCW)
in response to these allosteric effectors:
Activated by ATP
feedback inhibition by
cytidine triphosphate (CTP) & uridine triphosphate
ATCase Structure
R Subunit?
C Subunit?
R = Regulatory
is the HIGH activity conformation, more OPEN shape
C = Catalytic
Zinc Ion for Catalysis
What ACTIVATES ATCase?
What State is it at?
ATP activates ATCase
so when there is a abundance in fuel –> ATCase is active
R-STATE dominates
What INACTIVATES
ATCase?
T-STATE dominates
CTP** & **UTP
are end products of ATCase = Negative Feedback
when ASP is relatively low because it is being used up by ATCase
Once there is more ASP –> activate ATCase
OVERCOME the inhibition –> R-State
A-Subunit
trimeric structure of AMPK
KINASE activity
site of Phosphorylation
B-subunit
trimeric structure of AMPK
connector between alpha / gamma
also binds GLYCOGEN
may help in intracellular distribution of the complex
Y-Subunit
trimeric structure of AMPK
binds AMP / ATP
sensor of energy change
- *Assumptions for the MWC model**
- *All-or-none** model
R = Relaxed
(more active in catalysis / binding)
NO change in Catalytic ability for either T or R conformation
Vmax stays THE SAME
Allow for change in affinity as enzyme changes conformation
Km will CHANGE
will cause NON-linear Lineweaver-Burk Plots
Cooperativity in a Double Recip- Plot
Like Lineweaver-Burk
normally lineweaver-burk would ALLOW for us to find Km / Vmax
But a Non-linear plot indicates that the
M-M model is NOT Applicable
Positive Cooperativity –> CURVES UP
2 Main Causes for NON-LINEARITY
MORE Intermediates vs the M-M model
or
COOPERATIVITY
MWC Model & Double-Reciprocal Plot
MWC = Concerted
- *Slope = Km / Vmax**
- assuming that Vmax does NOT change*
Km does CHANGE due to changes in Enzyme Conformation
therefore the SLOPE will CHANGE as [S] changes
NON-LINEAR
Positive Cooperativity
Double Reciprical Plot
Binding of FIRST molecule ENHANCES the binding of the SECOND molecule
As [S] RISES –> the apparent affinity for S also RISES
indicating that Km will decrease –> slope of PLOT decreases too
since we need less [S] in order to reach 1/2 Vmax
Since it’s 1/[S] we look at it INVERSELY
CONCAVE UP CURVE
NEGATIVE Cooperativity
Double Reciprical Plot
Binding of FIRST molecule DECREASES the binding of the SECOND molecule
As [S] RISES –> the apparent affinity for S decreases
indicating that Km will INCREASE –> slope of the plot INCREASES
since we need MORE [S] in order to reach 1/2 Vmax
Since it’s 1/[S] we look at it INVERSELY
Concave DOWN
ATCase Mechanism
Carbamoyl Phosphate (CP) –> ASPartate
binding of CP INDUCES the enzyme to change its shape slightly
to Enhance Affinity for CP in the pocket
the shape change also creates a binding site for ASP, not there before
- *ASP** is oriented for Alpha-amino group to make a
- *NU attack** –> Carbonyl of CP
T-form –> R–form
