Enzyme Activity Assays

Enzyme activities were determined using modifications of the protocols of Moorhead and Plaxton (1988) for HK, FK, PFK, and PK, the protocols of Burrell et al. (1994) for G6PI, aldolase (ALD), triose phosphate isomerase (TPI), phosphoglycerate mutase (PGlyM), phosphoglycerate kinase (PGK), enolase (ENO), pyrophosphate-dependent phosphofructokinase (PFP), and UDP-glucose pyrophosphorylase (UDPase), the protocol of Plaxton (1990) for glyceraldehyde 3-phosphate isomerase (GAPDH), the protocol of Lu et al. (2014) for PEPase, and the protocols of Manjunath et al. (1998) and Davies et al. (2003) for phosphoglucomutase (PGM). Assays were performed at 25°C and coupled enzyme activity with NADH or NAD⁺ formation. NADH formation or consumption was measured at 340 nm using a SpectraMAX Plus microplate spectrophotometer (Molecular Devices Corp., Sunnyvale, CA, United States). Assay components were as follows. HK: 125 mM HEPES-NaOH (pH 7.5), 10 mM MgCl₂, 7 mM glucose, 1.5 mM NAD⁺, 2 U mL^-1 glucose 6-phosphate dehydrogenase (G6P-DH), and 0.25 mM ATP; FK: 125 mM HEPES-NaOH (pH 7.5), 10 mM MgCl₂, 3 mM fructose, 1.5 mM NAD⁺, 2 U mL^-1 G6P-DH, 6 U mL^-1 phosphoglucose isomerase, and 0.25 mM ATP; PFK: 50 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 2 mM EDTA, 2 mM fructose 6-phosphate, 0.1 mM NADH, 2 U mL^-1 ALD, 2 U mL^-1 TPI, 5 U mL^-1 glycerol 3-phosphate dehydrogenase, and 0.12 mM ATP; PK: 50 mM HEPES-NaOH (pH 7.0), 50 mM KCl, 10 mM MgCl₂, 2 mM DTT, 0.4 mg mL^-1 BSA, 1 mM phosphoenolpyruvate, 0.075 mM NADH, 26 U mL^-1 lactate dehydrogenase, and 1 mM ADP; PGM: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 30 μM glucose 1,6-bisphosphate, 0.5 mM NAD⁺, 2 U mL^-1 G6P-DH, and 0.9 mM glucose 1-phosphate (G1P); G6PI: 75 mM glycyl-glycine (pH 8.5), 10 mM MgCl₂, 1 mM NAD⁺, 1 mM fructose 6-phosphate, 0.5 U mL^-1 G6P-DH; ALD: 40 mM HEPES-NaOH (pH 7.7), 0.1 mM NADH, 5 mM fructose 1,6-bisphosphate (F1,6P), 1.7 U mL^-1 glycerol 3-phosphate dehydrogenase, 17 U mL^-1 TPI; TPI: 100 mM HEPES-NaOH (pH 8.0), 5 mM EDTA, 0.2 mM NADH, 1.5 mM DL-glyceraldehyde 3-phosphate, and 1 U mL^-1 glycerol 3-phosphate dehydrogenase; PGK: 100 mM HEPES-NaOH (pH 7.6), 1 mM EDTA, 2 mM MgSO₄, 0.2 mM NADH, 6.5 mM 3-phosphoglycerate, 1 mM ATP, and 3.3 U mL^-1 glycerol 3-phosphate dehydrogenase; PGlyM: 100 mM Tris-HCl (pH 7.6), 10 mM MgSO₄, 2.7 mM ADP, 0.2 mM NADH, 3 mM 3-phosphoglycerate, 1 U mL^-1 ENO, 5 U mL^-1 PK, 6 U mL^-1 lactate dehydrogenase, and 50 mM 3-phosphoglycerate; ENO: 100 mM HEPES-NaOH (pH 7.5), 10 mM MgCl₂, 1 mM NADH, 2.7 mM ADP, 0.5 mM 2-phosphoglycerate, 5 U mL^-1 PK, 6 U mL^-1 lactate dehydrogenase, and 10 mM 2-phosphoglycerate; PEPase: 50 mM Tris-HCl (pH 7.5), 1 mM phosphoenolpyruvate, 4 mM MgCl₂, 0.2 mM NADH, and 3 U lactate dehydrogenase; PFP: 100 mM Tris-HCl (pH 8.0), 5 mM fructose 6-phosphate, 2 mM sodium pyrophosphate, 5 mM MgCl₂, 0.20 mM NADH, 1 U mL^-1 ALD, 1.3 U mL^-1 glycerol 3-phosphate dehydrogenase, and 10 U mL^-1 TPI; UDPase: 100 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 1.6 mM NAD⁺, 0.8 mM UDP-glucose (UDPG), 4 U mL^-1 PGM, 4 U mL^-1 G6P-DH, and 0.4 mM sodium pyrophosphate; GAPDH: 100 mM Tris-HCl (pH 7.8), 4.5 mM 3-phosphoglycerate, 8 mM MgSO₄, 0.32 mM NADH, 2 mM ATP, 1 mM EDTA, 2 mM DTT, and 1.8 U mL^-1 PGK. Reactions were initiated by addition of ATP for HK, FK, and PFK assays; ADP for PK assay; G1P for PGM assay; 3-phosphoglycerate for PGlyM assay; 2-phosphoglycerate for ENO assay; sodium pyrophosphate for UDPase assay; and protein extract for G6PI, ALD, TPI, PGK, PEPase, PFP, and GAPDH assays. Additional details for enzyme assay protocols are available in Supplementary Table S1.