Figure 4: Asymmetric hybrid enzymes exhibit non-cooperative kinetics in the different reaction steps.

(a) Schematic representation of the created hybrids (covalent heterotrimers). Blue, red and yellow spheres represent dUTPase protomers containing the D102N, A98F and T148STOP mutations, respectively. Note, that all protomers contain the F158W mutation as well, except for the T148STOP mutant. Grey areas indicate enzymatic inactivity. (b) Steady-state kinetics of human dUTPase constructs: WWW (solid square), WWN (solid triangles), WNN (open triangles), WWF (solid stars), WWS (solid circle). Smooth lines through the data are hyperbolic fits yielding V_max = 7.9 ± 0.5 s⁻¹ for WWW, V_max = 4.4 ± 0.2 s⁻¹ for WWF, V_max = 3.8 ± 0.2 s⁻¹ for WWN, V_max = 1.8 ± 0.1 s⁻¹ for WNN, V_max = 2.9 ± 0.3 s⁻¹ for WWS. K_M values are listed in Table 1. (c) Comparison of the catalytic constants (striped bar) and apparent catalytic constants (grey bar) for determined by single turnover (transient kinetics) and steady-state experiments, respectively. See also Table 1 for the data. (d) Fluorescence intensity titrations upon dUTP binding to the various dUTPase constructs measured by stopped-flow (the symbol code is identical to that in panel (b). Smooth lines through data are hyperbolic fits yielding K_d values summarized in Table 1.