Summary /

The mode of action for the probe versus hPykM2 is assessed through analysis of the steady-state kinetics of PEP and ADP by the hPykM2. Fructose-1,6-bisphosphate is known to allosterically activate hPykM2 through induction of an enzyme state with a high affinity for PEP. In the absence of any of these activators, hPyk shows low affinity for PEP (KM ~ 1.5mM). In the presence of NCGC00031955 (CID_654376, SID_851783) or NCGC00030335 (CID_650361, SID_847943) or FBP, the KM for PEP decreased 10-fold to 0.13±0.04 mM or 0.1±0.02 mM for the two activators, respectively, with lesser effects on Vmax (values of 245 pmols/min with or without FBP and 255 pmols/min with NCGC00031955: CID_654376, SID_851783). In contrast, variation of the concentration of ADP in the presence and absence of activators shows that the steady-state kinetics are not significantly affected (KM for ADP = 0.1mM in either condition). Thus, NCGC00031955 (CID_654376, SID_851783) and NCGC00030335 (CID_650361, SID_847943) activate hPykM2by increasing the enzyme’s affinity for PEP and have little effect on ADP kinetics. This is similar to what we observed for FBP, which agrees with previous reports demonstrating increased affinity for PEP as the reason for activation of hPykM2 by FBP.