HPr and Adk activity was measured by performing HPr-P-dependent allosteric inhibition (when a ligand - a molecule or ion - binds to a site, other than the active site, to inhibit activity) through a coupled assay involving PEP and LDH. 1 uM of HPr or HPr-P was added to an assay mixture. In this experiment, HPr-P had an effect. HPr-P was then incubated for 40 minutes at 30 degrees Celsius. In a separate reaction mixture, 1 ng of Adk was added to 100 uL of a kinase reaction mixture containing several concentrations of substances including 0-2 mM of AMP, 1.2 mM ATP, 1.2 mM PEP, .3 mM NADH, 1.2 units of PK, and 1.2 units of LDH. 1 uM HPr-P from the first assay mixture was then added into the Adk reaction mixture. The PEP was converted to pyruvate by PK in an ADP-dependent enzymatic reaction. The pyruvate was used as the substrate and converted to lactate by LDH in a second enzymatic reaction in the presence (+HPr) or absence (-HPr) of HPr. During the second enzymatic reaction, the NADH was oxidized to NAD+; the NADh to NAD+ oxidation concentration in the presence and absence of HPr-P was measured to give a graph of the steady-state kinetics (when all state variables are constant; everything but the varying concentrations of AMP). Steady state kinetics include the Khalf, Vmax, and the Hill Coefficient, shown in Table 4.

In Figure 4, the x-axis shows the varying AMP concentration in mM, and the y-axis shows the activity % of Adk. Two slopes are present, the one with +HPr-P is with presence and the one with -HPr-P is absence. The slope with -HPr-P shows a quick % increase in activity, reaching close to 1%, while +Hpr-P shows a slower activity % rise to around 1% as well, showing the inhibition when HPr-P is present. The Vmax of +HPr-P is around 936 U/mg, while the Vmax around -HPr-P is around 736 U/mg.

Graph 4. Allosteric inhibition of Adk by HPr-P. Graph shows Adk activity % dependent on varying AMP concentration and absence or presence of HPr-P. The presence of HPr-P is identified by +HPr-P and the absence of HPr-P is identified by -HPr-P.

Adapted from "The phosphocarrier protein HPr of the bacterial phosphotransferase system globally regulates energy metabolism by directly interacting with multiple enzymes in Escherichia coli." by Rodionova IA, Zhang Z, Mehla J, Goodacre N, Babu M, Emili A, Uetz P, Saier MH Jr., 2017, J Biol Chem, Page 14253.

Table 4. Steady State Kinetics of Adk in the absence (-HPr-P) and presence (+HPr-P) of HPr-P. Table shows the Vmax, Hill Coefficient, and Khalf of the activity. They are visually represented in Graph 4.

Adapted from "The phosphocarrier protein HPr of the bacterial phosphotransferase system globally regulates energy metabolism by directly interacting with multiple enzymes in Escherichia coli." by Rodionova IA, Zhang Z, Mehla J, Goodacre N, Babu M, Emili A, Uetz P, Saier MH Jr., 2017, J Biol Chem, Page 14252.

Figure 4. Complex of HPr and Adk. Figure shows the complex of HPr and Adk. HPr is represented by green and Adk is represented by purple.

Adapted from "The phosphocarrier protein HPr of the bacterial phosphotransferase system globally regulates energy metabolism by directly interacting with multiple enzymes in Escherichia coli." by Rodionova IA, Zhang Z, Mehla J, Goodacre N, Babu M, Emili A, Uetz P, Saier MH Jr., 2017, J Biol Chem, Page 14254.