2 phosphoglycerate 2 pg

ENO1 activity was measured in the reaction buffer containing 50 mmol/L Tris‐HCl (PH 7.5), 100 mmol/L KCl, 25 mmol/L MgSO4, 1.3 mmol/L ADP (Sigma, MO, USA), and 0.15 mmol/L nicotinamide adenine dinucleotide (NADH, Sigma, MO, USA). In addition, 0.6–1 U of pyruvate kinase and 0.9–1.4 U of lactate dehydrogenase (Sigma, MO, USA) were added to the reaction solution. After adding 1.9 mmol/L 2‐phosphoglycerate (2‐PG, Shanghai yuanye Bio‐Technology, Shanghai, China) and appropriate amount of ENO1 (1 μg of purified rENO1 or 10 μg of cell lysates), the absorbance in 340 nm as a measure of NADH was recorded at room temperature every 1 min for 10−30 min on a Microplate Photometer (Thermo Fisher Scientific, MA, USA).

Enolase activity was determined by direct spectrophotometric assay via measuring the increase of absorbance at 240 nm of phosphoenolpyruvate (PEP) as described previously with some modifications [62 (link)]. Briefly, the reaction buffer (pH 7.0) containing 10 mM imidazole, 200 mM KCl, and 0.5 mM MgAc in a final reaction volume of 100 mL was mixed with Eno1 (a final concentration of 30 nM), followed by the mixture of 2-phosphoglycerate (2-PG) (Yuanye Biotech Shanghai, China) with a final concentration of 1 mM. The enolase activity of Eno1 was evaluated by measuring the increase of absorbance (OD240) at room temperature for 10 min. For the inhibition study of enolase by compounds, compounds at a concentration of 200 μM were used to mix well with Eno1 and were incubated at room temperature for 5 min, and subsequent operations were described above.