31 - Allosterism & Cooperativity Flashcards

1
Q

Allosterism Definition

A

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

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2
Q

Allosteric Effectors

A

LIGAND for an allosteric protein

  • *Small molecules** that modulate the conformation & activity of:
  • *Enzymes** / Receptors / Proteins
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3
Q

What is the purpose of ALLOSTERISM?

A

to alter the overall activity
changes at 1 site –> affect the entire assembly / DRASTIC changes

PROMOTE SENSITIVITY
to changing cellular conditions, an Evolutionary advantage

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4
Q

What is COOPERATIVITY?

A

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

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5
Q

POSITIVE Cooperativity

A

when the binding of 1 ligand

AUGMENTS** or **INCREASES

the binding of the second molecule

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6
Q

Negative Cooperativity

A

when the binding of the 1st ligand

REDUCES

binding by the 2nd ligand

NOT inhibition, just reduces the binding

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7
Q

Hemoglobin & O2 Binding

A

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

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8
Q

Heme Oxygenation

A

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

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9
Q

T State of Hemoglobin

A
  • 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

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10
Q

R-State of Hemoglobin

A

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**
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11
Q

BOHR EFFECT

A

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

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12
Q

MCW Model

one of 2 Models for combining Allosterism & Cooperativity

A

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

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13
Q

Explaination of the MCW Model

one of 2 Models for combining ​Allosterism & Cooperativity

A

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

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14
Q

KNF Model

one of 2 Models for combining ​Allosterism & Cooperativity

A

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

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15
Q

Which model for

Allosterism & Cooperativity

Is the DIAGONAL / VERTICAL?

A

Diagonal = KNF
Induced Fit / Sequential Model

Vertical = MWC
All-or-none shape change

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16
Q

ATCase Function

A

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

17
Q

ATCase Structure

R Subunit?

C Subunit?

A

R = Regulatory
is the HIGH activity conformation, more OPEN shape

C = Catalytic

Zinc Ion for Catalysis

18
Q

What ACTIVATES ATCase?
What State is it at?

A

ATP activates ATCase
so when there is a abundance in fuel –> ATCase is active

R-STATE dominates

19
Q

What INACTIVATES
ATCase?

A

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

20
Q

A-Subunit

trimeric structure of AMPK

A

KINASE activity

site of Phosphorylation

21
Q

B-subunit

trimeric structure of AMPK

A

connector between alpha / gamma

also binds GLYCOGEN

may help in intracellular distribution of the complex

22
Q

Y-Subunit

trimeric structure of AMPK

A

binds AMP / ATP

sensor of energy change

23
Q
  • *Assumptions for the MWC model**
  • *All-or-none** model

R = Relaxed
(more active
in catalysis / binding)

A

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

24
Q

Cooperativity in a Double Recip- Plot
Like Lineweaver-Burk

A

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

25
Q

2 Main Causes for NON-LINEARITY

A

MORE Intermediates vs the M-M model

or

COOPERATIVITY

26
Q

MWC Model & Double-Reciprocal Plot

A

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

27
Q

Positive Cooperativity

Double Reciprical Plot

Binding of FIRST molecule ENHANCES the binding of the SECOND molecule

A

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

28
Q

NEGATIVE Cooperativity

Double Reciprical Plot

Binding of FIRST molecule DECREASES the binding of the SECOND molecule

A

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

29
Q

ATCase Mechanism

A

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

30
Q
A