Skip to main content
. 2020 Apr 7;9:e56418. doi: 10.7554/eLife.56418

Figure 4. Effect of S416D phosphomimetic substitution on enzymatic activity.

(A) Main splice variants of hIMPDH1: hIMPDH1α (546aa) and hIMPDH1γ (595aa) contain extended sequences at the COOH- terminus (546aa) or both the NH2- and COOH-termini (595aa). (B) S416D mutant recombinant proteins were rigorously normalized to their wildtype counterparts preceding enzymatic analysis. (C) Effect of S416D substitution on the Michaelis-Menten kinetics of hIMPDH1β (514aa); hIMPDH1α (546aa); and hIMPDH1γ (595aa) isoforms. Mutation S416D increased the Km for IMP and decreased the Vmax significantly in hIMPDHα (546aa) and γ (595aa) splice variants, with the effect being maximal in the hIMPDH1α (546aa) isoform. Kinetic parameters were: hIMPDH1β (514aa) [Vmax = 0.099 ± 0.004, Km = 7.62 ± 1.69]; S416D/hIMPDH1β (514aa) [Vmax = 0.084 ± 0.002, Km = 19.13 ± 1.75]; hIMPDH1α (546aa) [Vmax = 0.152 ± 0.002, Km = 20.72 ± 1.32]; S416D/hIMPDH1α (546aa) [Vmax = 0.046 ± 0.002, Km = 59.59 ± 9.74]; hIMPDH1γ (595aa) [Vmax = 0.093 ± 0.003, Km = 12.86 ± 2.18]; S416D/hIMPDH1γ (595aa) [Vmax = 0.049 ± 0.001, Km = 13.95 ± 1.71]. Results represent the media and S.E.M of three independent experiments with three technical replicates each.

Figure 4.

Figure 4—figure supplement 1. Effect of phosphomimetic substitutions on enzymatic activity.

Figure 4—figure supplement 1.

(A) Reaction kinetics of wildtype; S160D; S416D and S477D mutants of hIMPDH1α (546aa). Assays were performed with 3,6 μM of recombinant protein in assay buffer with 125 mM IMP and 0.5 mM NAD+. Results represent the mean and S.E.M of three independent experiments with three technical replicates each. S160D and S477D mutants showed wildtype kinetics; while S416D mutant showed delayed kinetics. (B) Michaelis-Menten plots for the wildtype and phosphomimetic mutants. NADH production plotted to concentration of IMP. Assays were performed at 0.5 mM NAD+ and varying concentrations of IMP, from 0 to 500 μM. NADH production (nmol/min/mg) was obtained from ΔAbs/Δt by applying Lambert- Beer’s law (εNADH340nm=6220M−1cm−1, l = 0.58 cm), and results were fitted to Michaelis-Menten curves with GraphPad Prism [Mean and S.E.M of three independent experiments, three technical replicas each].