The figure on the right is a Lineweaver-Burk plot of P. furiosus PFK with [fruct
ID: 767348 • Letter: T
Question
The figure on the right is a Lineweaver-Burk plot of P. furiosus PFK with [fructose-6-P] held constant and [ADP] as the variable substrate (): in the presence of 5mM ATP (0): or in the presence of 5 mM AMP (X; Tuininga et al., 1999). How does the addition of ATP affect PFK affinity for ADP? How does the addition of AMP affect PFK affinity for ADP? What do these data indicate about the role(s) of ATP: ADP: and AMP in PFK activity in P. furiosus? Compare and contrast the P. furiosus results with what is known about the roles of ATP: ADP: and AMP in regulating glycolysis in eukaryotes.Explanation / Answer
a. How does the addition of ATP affect PFK affinity for ADP ?
It was demonstrated that as phosphoryl group donor, ADP could be replaced by GDP, ATP, and GTP to a limited extent. Further, the literature suggests that ATP can act as competitive inhibitor of ADP-dependent phosphofructokinase. Because of this the addition of ATP may reduce the affinity of PFK to ADP as it competes with binding of ADP in catalytic site.
b. How does the addition of ATP affect PFK affinity for ADP ?
AMP was also identified as one of the competitive inhibitor of the phosphofructokinase. Addition of excess AMP will reduce the PFK affinity for ADP.
c. What do these data indicate the role(s) of ATP, ADP and AMP in PFK activity in P. furiosus?
Phosphofructokinase is ADP dependent for transfer of phosphoryl group. ADP is its main substrate for transfer of phoshoryl grou. To some extent ATP can act as phophoryl donor as well which leads it to act as competitive inhibitor of ADP. Besides ATP, the PFK is also allosterically inhibited by AMP as well.
d. Compare and contrast the P. furiosus results with what is known about the roles of ATP, ADP and AMP in regulating glycolysis in eukaryotes?
Phosphofructokinase from P. furiosus was found to be inhibited by ATP and AMP through a competitive mechanism. In the case of ATP, this is not surprising, because ATP itself is a substrate and however it is surprising to see that ATP and AMP have the same (negative) effect on the activity of the phosphofructokinase. Allosterically regulated phosphofructokinases are usually inhibited by ATP but stimulated by AMP. Both yeast and mammalian phosphofructokinases are regulated by a large variety of effectors. Only mammalian enzymes are allosterically activated by fructose 1,6-bisphosphate. A very potent allosteric stimulator of eukaryotic phosphofructokinases is fructose 2,6-bisphosphate, which acts synergistically with AMP and is detected in most eukaryotes but never in prokaryotes. Apparently, the ADP-dependent phosphofructokinase from P. furiosus is not allosterically regulated at all, and therefore it can not act as the major control point of the glycolytic pathway. However, the glyceraldehyde-3-phosphate ferredoxin oxidoreductase could be an important enzyme in control of the glycolysis of P. furiosus.
Reference: http://www.jbc.org/content/274/30/21023.full.pdf