Extended Data Fig. 5: Enzyme kinetics of hexokinases with and without recombinant KRAS4A and with and without 2-DG. | Nature

Extended Data Fig. 5: Enzyme kinetics of hexokinases with and without recombinant KRAS4A and with and without 2-DG.

From: KRAS4A directly regulates hexokinase 1

Extended Data Fig. 5: Enzyme kinetics of hexokinases with and without recombinant KRAS4A and with and without 2-DG.

a, The activity of recombinant full-length HK1 is unaffected by recombinant KRAS4A. Reaction velocity is plotted (mean ± s.e.m.) as a function of glucose concentration. Velocities are plotted with or without the addition of recombinant, GTP-loaded KRAS4A or RAC2. Plots combine independent assays (n = 4). b, Enzyme kinetics of hexokinases. Full-length HK1 and HK2 and the catalytic C-terminal domain of HK1 were expressed in E. coli as GST fusion proteins and affinity purified with glutathione–agarose beads. Hexokinase activity on the beads was measured with a linked assay kit (BioVision) in which the glucose-6-phosphate produced is oxidized by glucose-6-phosphate dehydrogenase to form NADH, which reduces a colourless probe to a coloured product with strong absorbance at 450 nm. Vmax and Km were calculated by nonlinear regression using GraphPad Prism v 8.1.1 and the goodness of fit is given as R2.

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