PFK Flashcards
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What does PFK stand for?
Phosphofructokinase
What kind of a molecule is PFK
a regulatory enzyme
Which pathway is PFK key in? And which step does it function in?
Glycolytic pathway which is the breakdown of glucose to pyruvate
What is PFK inhibited/activated by?
inhibited allosterically by one of the end products of the pathway - PHOSPHOENOLPYRUVATE. activated by rising levels of ADP. (also inhibited by phosphoglycolate)
Which stage of the pathway is PFK involved with?
it catalyzes phosphorylation by ATP of fructose phosphate to give f-1,6-bp
Bacterial PFK is multimeric. What does this mean?
It means there are 4 identical subunits, each of 320 amino acids
Describe allosteric protein states
Allosteric proteins exist in 2 states, R for relaxed and T for tense. BINDING OCCURS MORE EASILY IN THE RELAXED STATE.
What is the rule between effector molecules and affinity for allosteric proteins?
Effector molecules have a high affinity for only ONE of allosteric proteins states.
What are effector molecules?
They are small molecules that bind to a place distance from the active site of an enzyme, in this case, and regulate its biological activity.
Which kind of effector molecules (positive or negative) are activators/inhibitors and which of the 2 allosteric states of PFK do they bind to?
positive effectors are ACTIVATORS and negative effectors are INHIBITORS. + binds to T state and - binds to R state. All affect binding of PFK for f6p.
What is significant about the binding of ATP?
Non-cooperative
What shape/size molecule is PFK in humans?
It is a tetramer with 2 domains, one small and one large, each a beta barrel with a cylindrical beta sheet surrounded by alpha helices - a rossman fold
How are the subunits of PFK linked?
Pairswise, AB and CD. AB is the REAL dimer.
What is a rossman fold?
a beta barrel with a cylindrical beta sheet surrounded by alpha helices
Which is the ‘real’ dimer?
AB
How do the A-D subunits connect?
A-D connects through a small interface where allosteric changes occur!
How do the A-B subunits connect?
AB connect via the LARGE interface where no real changes occur!
What is the relationship between the small interface contacts and the allosteric states of PFK?
They differ, a rotation of around 7degrees. This leads to changes in the detail of the tertiary structure - so changes in the affinity for F6P. LARGE INTERFACE dimer contacts remain unchanged.
Go into detail about the T state of allostery.
The t-tense-state has an inhibitor bound at the effector site and is INACTIVE. This is because the small contact changes have led to changes in the tertiary structure which brings the dimers closer together, H-bonds forming between dimers across the small interface.
Go into detail about the R state of allostery
The r-relaxed-state has ADP bound at the effector site; an activator; and is ACTIVE. The changes have led to the two loops/domains being further apart and water molecules enter the space.
How many binding sites does each domain have?
3
Where are the f6p and ATP binding domains?
together between 2 domains, large domain binds ATP whilst f6p is bound by both domains. phosphate ALSO interacts with a neighboring subunit across the small interface
Where is the regulatory effector site?
Distant from the active site, lying at the dimer-dimer interface
What molecule interacts with F6P in the R-state?
F6P interacts with an arginine residue on 6F loop in a neighboring subunit in the other dimer, this is CRUCIAL FOR ACTIVITY.
What is the regulatory site at the dimer dimer interface linked to?
Linked to F6P in the OTHER dimer through Falpha helix
What structural changes occur to reduce affinity whilst in the T state?
The F-helix partially unwinds so Arg162 points away from F6P and glu161 points towards - F6P is repelled and affinity reduces 1000 fold
SO IN SUMMARY what is allostery dependant on?
relative conc of ADP and PEP (phosphoenolpyruvate)
