Figure 1.
Pathway context of GlmU-catalyzed reactions.
Figure 2.
Equilibria between enzyme species for ordered bi-bi mechanism of enzymatic reaction.
A, B = First and second substrate of enzyme E; P, Q = First and second substrates for the reverse reaction, their binding to enzyme accounts for product inhibition; I = Different types of hypothetical inhibitor, whose type is determined by the form of enzyme it binds to: I binding to free E (forming E-I complex) is a competitive inhibitor with respect to A, I binding to E-A complex (forming E-A-I complex) is uncompetitive inhibitor with respect to A and I binding to E-A-B complex (forming E-A-B-I complex) is uncompetitive inhibitor with respect to both A and B; Kic = Inhibition constant of hypothetical competitive inhibitor; Kiu_<metabolite> = Inhibition constant of hypothetical uncompetitive inhibitor where the inhibitor behaves uncompetitive against the metabolite indicated within <>.
Table 1.
Kinetic parameters used for simulating the model.
Table 2.
Initial metabolite concentrations and the boundary conditions for various variants of model.
Figure 3.
Experimental vs. simulated concentration response curves.
GlcNAc1P concentration response curve; Curves obtained from experiment: Black; Curves obtained from simulation: Gray; v = Velocity of GlmU rxn-2. Assays were carried out at 25°C in assay buffer containing 50 mM Hepes KOH pH 7.5, 5 mM MgCl2. 5 mM DTT, 0.3 units/ml pyrophosphatase and the phosphate formed was detected using malachite green reagent from Innova Biosciences. For GlcNAc1P KM determination UTP was fixed at 250 µM.
Figure 4.
Dynamic behaviour of the rates of GlmU reactions in coupled vs. decoupled models.
Plots corresponding to medium ( = KM) metabolite concentrations; v = Rates of GlmU rxn-1 (broken lines) and rxn-2 (solid lines); Panel 1A: In vitro variant GlmU rxn-1; Panel 1B: In vitro variant GlmU rxn-2; Panel 2A: In vivo variant GlmU rxn-1; Panel 2B: In vivo variant GlmU rxn-2; Coupled model: Black lines; Decoupled model: Gray lines.
Figure 5.
Effect of in silico inhibition of GlmU reactions under various conditions.
Metabolite concentrations used for simulation: Low ( = 0.1xKM), Medium ( = KM), High ( = 10xKM) and Intracellular levels; Inhibition strength (I/Ki ratio) maintained at 20; Numbers in the figure indicate percent decrement in GlmU overall rate due to various types of inhibition; Linear color-coded scale from Gray to White indicating decreasing level of effect of inhibition on GlmU rate).
Figure 6.
Dynamics of GlcNAc1P (black line) and UTP (gray line) normalised concentrations under the proposed assay condition.
The normalized concentrations of both GlcNAc1P and UTP stay above 1 for significant portion of simulation time period, which is a favourable condition of assay for identifying uncompetitive (against E-UTP-GlcNAc1P complex) inhibitors against GlmU rxn-2.