Biochemistry I - Regulation of PFK by F26P (PFK-DL2)

In the following dry lab you will measure the activity of PFK as a function of F-6-P concentration in the absense or presence of various amounts of F26P. The concentration of ATP and AMP (Activator) are set to give the optimal activity for the enzyme - you will adjust only the F-6-P and F26P levels.

  1. Before starting, review the reaction catalyzed by PFK in glycolysis. What are the substrates and what are the products?
  2. How does the structure of the regulatory compound, F26P, differ from the substrates and products for PFK?
  3. Vary [F6P] and record the initial velocity of PFK in the absence of F26P(left box, yellow) and in the presence of 25 uM F26P (right box, orange).
  4. How does F26P affect the reaction velocity?
  5. Where do you think F26P binds to the enzyme, in the active site, or elsewhere?
  6. Use the effect on the velocity (F6P saturating) to measure the Kd for F26P binding.
1. Enter values for [F6P] in the left box, ranging from 1 to 100 uM (e.g. 1, 5, 10, 20 ,50, 100, the Km is 10 um).
 a) Use [F26P]=25 uM to evaluate whether it is an inhibitor or activator of PFK.
[F6P] = uM [F26P] = uM
2. For the above values of F6P and F26P, Calculate vi
vi = uM/min([F26P]=0) vi = uM/min ([F26P] Present)
(Each calculated vo value has a small "experimental error" added to it.)