Enzyme regulation is an important process in controlling metabolic flux. The two ways in which this is thought to happen is by inhibiting an enzyme with a small molecule or through a covalently-bound post-translational modification. This second process is thought to require an enzyme to form this covalent bond. Moellering and Cravatt challenge this paradigm by showing that inherently reactive intermediates can form covalent bond modificatins without the use of an enzyme. They do this using 1,3-bisphosphoglycerate (1,3-BPG), which is formed by the enzyme GAPDH in the process of glycolysis. This intermediate was thought to be a prime candidate for study because its acylphosphate group makes it inherently reactive, especially with lysine residues. If 1,3-BPG reacts with lysine, it would form 3-phosphoglyceryl-lysine (pgK).
The authors began by combining GAPDH with its substrate and cofactor to produce 1,3-BPG. GAPDH was then separated using LC/MS, and they found that GAPDH was, indeed, modified to form pgK residues. To determine the scope of 1,3-BPG modification, the authors incubated cells with a high concentration of glucose. They found that there were a greater number of pgK residues in several glycolytic enzymes, including enolase 1, relative to control cells.
Now that the authors determined that pgK residues can form without the use of an enzyme and that it can be found on multiple enzymes, they sought to show that these pgK-modified proteins will be inactivated. They performed an in vitro study where 1,3-BPG was removed by dialysis. This showed a lower Km than when 1,3-BPG remained in solution. Therefore, Moellering and Cravatt demonstrate that when 1,3-BPG is at high levels, it will turn off enzymes by forming covalent bonds without the aid of another enzyme. This offers a new method by which enzymes can be modified and, therefore, regulated.