PFK

Question 1

What does PFK stand for?

Answer 1

Phosphofructokinase

Question 2

What kind of a molecule is PFK

Answer 2

a regulatory enzyme

Question 3

Which pathway is PFK key in? And which step does it function in?

Answer 3

Glycolytic pathway which is the breakdown of glucose to pyruvate

Question 4

What is PFK inhibited/activated by?

Answer 4

inhibited allosterically by one of the end products of the pathway - PHOSPHOENOLPYRUVATE. activated by rising levels of ADP. (also inhibited by phosphoglycolate)

Question 5

Which stage of the pathway is PFK involved with?

Answer 5

it catalyzes phosphorylation by ATP of fructose phosphate to give f-1,6-bp

Question 6

Bacterial PFK is multimeric. What does this mean?

Answer 6

It means there are 4 identical subunits, each of 320 amino acids

Question 7

Describe allosteric protein states

Answer 7

Allosteric proteins exist in 2 states, R for relaxed and T for tense. BINDING OCCURS MORE EASILY IN THE RELAXED STATE.

Question 8

What is the rule between effector molecules and affinity for allosteric proteins?

Answer 8

Effector molecules have a high affinity for only ONE of allosteric proteins states.

Question 9

What are effector molecules?

Answer 9

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.

Question 10

Which kind of effector molecules (positive or negative) are activators/inhibitors and which of the 2 allosteric states of PFK do they bind to?

Answer 10

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.

Question 11

What is significant about the binding of ATP?

Answer 11

Non-cooperative

Question 12

What shape/size molecule is PFK in humans?

Answer 12

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

Question 13

How are the subunits of PFK linked?

Answer 13

Pairswise, AB and CD. AB is the REAL dimer.

Question 14

What is a rossman fold?

Answer 14

a beta barrel with a cylindrical beta sheet surrounded by alpha helices

Question 15

Which is the ‘real’ dimer?

Answer 15

AB

Question 16

How do the A-D subunits connect?

Answer 16

A-D connects through a small interface where allosteric changes occur!

Question 17

How do the A-B subunits connect?

Answer 17

AB connect via the LARGE interface where no real changes occur!

Question 18

What is the relationship between the small interface contacts and the allosteric states of PFK?

Answer 18

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.

Question 19

Go into detail about the T state of allostery.

Answer 19

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.

Question 20

Go into detail about the R state of allostery

Answer 20

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.

Question 21

How many binding sites does each domain have?

Answer 21

3

Question 22

Where are the f6p and ATP binding domains?

Answer 22

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

Question 23

Where is the regulatory effector site?

Answer 23

Distant from the active site, lying at the dimer-dimer interface

Question 24

What molecule interacts with F6P in the R-state?

Answer 24

F6P interacts with an arginine residue on 6F loop in a neighboring subunit in the other dimer, this is CRUCIAL FOR ACTIVITY.

Question 25

What is the regulatory site at the dimer dimer interface linked to?

Answer 25

Linked to F6P in the OTHER dimer through Falpha helix

Question 26

What structural changes occur to reduce affinity whilst in the T state?

Answer 26

The F-helix partially unwinds so Arg162 points away from F6P and glu161 points towards - F6P is repelled and affinity reduces 1000 fold

Question 27

SO IN SUMMARY what is allostery dependant on?

Answer 27

relative conc of ADP and PEP (phosphoenolpyruvate)