Extended Data Fig. 8: Functional analysis of wild-type pyruvate oxidase and wire variants.

a, b, Stopped-flow kinetic analysis of substrate binding in pyruvate oxidase catalysis using substrate analogue methylacetyl phosphonate (MAP). The analogue forms a covalent conjugate with bound cofactor ThDP but is not further processed, enabling the exclusive analysis of substrate binding. a, Selected stopped-flow transients monitored after mixing pyruvate oxidase (wild-type, E60Q, E60A, H89N and H89A variants) with 10 mM MAP. b, Dependence of the observed first-order rate constants k_app on the MAP concentration. Data were fitted with Supplementary Equation 10 to obtain estimates of the substrate dissociation first-order rate constant k_off (non-zero intercept with y-axis), substrate association second-order rate constant k_on (slope) and substrate dissociation equilibrium binding constant K_D^app (k_off/k_on). All measurements were carried out in triplicate and are shown as mean ± s.d. All experiments were independently repeated twice with similar results. Kinetic and thermodynamic constants are given in Extended Data Table 2. c–f, Stopped-flow kinetic analysis of substrate binding and processing in pyruvate oxidase catalysis under single turnover conditions. c, Stopped-flow transients obtained upon mixing wild-type pyruvate oxidase with various concentrations of substrate pyruvate under anaerobic conditions, using the intrinsic absorbance of the bound cofactor FAD. d, Dependence of the calculated first-order rate constants k_app on the applied pyruvate concentration for wild-type pyruvate oxidase. Data were fitted with Supplementary Equation 12. Inset, a magnified section of the graph showing the substrate dependence in the concentration range 0–10 mM. Note the sigmoidal dependence, which indicates positive cooperativity (Extended Data Table 2). e, Dependence of the first-order rate constants k_app on the applied pyruvate concentration for the E59Q variant. Data were fitted with Supplementary Equation 12. f, Dependence of the first-order rate constants k_app on the applied pyruvate concentration for the wild-type enzyme and the H89N, H89A, E60Q and E60A. Data were fitted with Supplementary Equation 12. All measurements were carried out in triplicate and are shown as mean ± s.d. All experiments were independently repeated twice with similar results. Kinetic and thermodynamic constants are given in Extended Data Table 2.