Phosphofructokinase (L3)

Question 1

What is the function of PFK?

Answer 1

The main regulatory enzyme in glycolysis

Question 2

What is PFK made up from?

Answer 2

4 identical subunits of 320 AA’s in an alpha-beta structure reminiscent of a Rossman Fold

Question 3

What is an allosteric control?

Answer 3

Regulation of an enzyme or other protein by binding an effector molecule at the protein’s allosteric site (that is, a site other than the protein’s active site)

Question 4

Name to two states in which an allosteric proteins exist.

Answer 4

T (Tense) state and R (Relaxed) State

Question 5

How are the dimers arranged in PFK?

Answer 5

A-B and C-D

Question 6

What is the degree of rotation between the R and T states?

Answer 6

7 Degrees

Question 7

What does the 7 degree shift achieve?

Answer 7

Changes in the tertiary structure leading to higher affinity for fructose-6-phosphate

Question 8

What bonds are formed in the inactive T state?

Answer 8

H bonds are formed between the dimers across the small interface due to their close proximity

Question 9

What bonds are formed in the active R state?

Answer 9

H bond bridges due to the loops being further apart and water being allowed to enter

Question 10

How many binding sites does each of the four subunits contain?

Answer 10

3

Question 11

Where does the binding site for ATP and F6P lie?

Answer 11

In the active site cleft between the two domains

Question 12

Which domain binds ATP?

Answer 12

Large Domain

Question 13

Which domain binds F6P?

Answer 13

Both domains, with the phosphate also interacting with a neighbouring subunit, across the small interface, in the other dimer

Question 14

Where is the third regulatory site?

Answer 14

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

Question 15

How is the 3rd regulatory site linked to the F6P site in the other dimer?

Answer 15

Through the F (150-160) alpha helix and the 161-162 turn at its C-terminus.

Question 16

Explain the interactions of the R state PFK

Answer 16

The F6P phosphate interacts with an Arginine residue (Arg162) on the 6-F loop (connecting strand 6 and helix F) in a neighbouring subunit in the other (not the same!!) dimer.

Question 17

Explain the interaction of the T state PFK

Answer 17

The subunit reorganisation leads to the F helix being partially unwound so that Arg162 points away from F6P and Glu161 points towards, and thus repels, the F6P phosphate and affinity for F6P is reduced 1000-fold.