Allosteric Regulators on Pyruvate Kinase Oxidation

Research has been going extremely well lately. The main focus of my research over the past month has been the role of fructose-1,6-bisphosphate in preventing oxidative damage to the enzyme of interest – pyruvate kinase. PK has two main binding sites with cysteines critical for function: the catalytic active site and the F-1,6-bisP regulatory site. Because both of these sites have cysteines (the amino acid we’re studying in our lab), they can be oxidized by HOCl and DTNB, which can affect their activity.

Earlier in this project, I analyzed the role of ATP in protecting against oxidation and found that the molecule is successful at minimizing thiol modification. (ATP is a regulator of PK because, as a product of the reaction PK catalyzes, ATP shifts the reaction in the reverse direction. The enzymatic requirements for PK to work in reverse, however, make it impossible – one of the three irreversible steps in glycolysis.) The thought was that ATP, which binds in the ADP/ATP catalytic active site and is stabilized by Mg2+ ions, could physically block HOCl molecules from interacting with the cysteines in the active site to limit oxidative damage. We were pleased to see this was the result.

With that same logic, we wanted to see if F-1,6-bisP can work in the same way. The research seems to indicate that F-1,6-bisP does exhibit protective effects on the enzyme, which is important because the regulation can add another layer to keep the enzyme functional despite a degree of oxidative conditions. Our main question from here is whether or not F-1,6-bisP could reverse oxidative damage in its binding site or if it is blocking HOCl from interacting with the cysteine’s purely because it increases the amount of “stuff” the oxidant has to maneuver around in order to reach the binding site. I’m going to wrap this up soon before heading into the tail end of this honor’s project.