Extended Data Fig. 6: Detection of cooperativity in human OMPDC catalysis.
Detection of cooperativity in human OMPDC catalysis. (a) Steady-state kinetic analysis of OMPDC-catalysed conversion of OMP into UMP by isothermal titration calorimetry showing the raw data thermogram after a single injection of 0.2 mM OMP to a solution containing 1 µM OMPDC in 20 mM HEPES/NaOH, pH 7.4 at 25 °C and the therefrom calculated v_S plot. The data were fitted with the Hill equation (fit shown in red). Note the estimated Hill coefficient of nH = 2.0 that indicates positive cooperativity between the two active sites in the homodimeric enzyme. All measurements were carried out in triplicate and are shown as mean ± s.d. (b) Thermodynamic analysis of binding of product UMP to human OMPDC using isothermal titration calorimetry showing the raw data thermogram and the integrated heats. The data were fit with a two-site binding model as detailed in the methods section (fit shown in red). The observation of two binding sites suggests a negative cooperativity between the two active sites in the homodimeric enzyme. All experiments were carried out as triplicates with almost identical results. The fitted kinetic and thermodynamic constants along with the associated calculated standard deviation are shown for a representative experiment. (c) Putative communication wires in human OMPCase that link the two remote active sites of the homodimer. Shown is the structure of human OMPDC variant Lys314AcLys in complex with substrate OMP highlighting the two active sites with the bound substrate molecules and two potential signaling pathways involving Asp317*-W1-W2-W3-W4-W5-Asp317 and/or His283-Glu311-Asp285 from both subunits and several water molecules. Abbreviations: OMPDC, orotidine-5’-monophosphate decarboxylase; OMP, orotidine-5’-monophosphate; UMP, uridine-5’-monophosphate.
