Reactivity of Toluate Dioxygenase with Substituted Benzoates and Dioxygen

Toluate dioxygenase (TADO) of Pseudomonas putida mt-2 catalyzes the dihydroxylation of a broad range of substituted benzoates. The two components of this enzyme were hyperexpressed and anaerobically purified. Reconstituted TADO had a specific activity of 3.8 U/mg with m-toluate, and each component had a full complement of their respective Fe(2)S(2) centers. Steady-state kinetics data obtained by using an oxygraph assay and by varying the toluate and dioxygen concentrations were analyzed by a compulsory order ternary complex mechanism. TADO had greatest specificity for m-toluate, displaying apparent parameters of KmA = 9 ± 1 μM, k(cat) = 3.9 ± 0.2 s(−1), and K(m)O(2) = 16 ± 2 μM (100 mM sodium phosphate, pH 7.0; 25°C), where K(m)O(2) represents the K(m) for O(2) and KmA represents the K(m) for the aromatic substrate. The enzyme utilized benzoates in the following order of specificity: m-toluate > benzoate ≃ 3-chlorobenzoate > p-toluate ≃ 4-chlorobenzoate ≫ o-toluate ≃ 2-chlorobenzoate. The transformation of each of the first five compounds was well coupled to O(2) utilization and yielded the corresponding 1,2-cis-dihydrodiol. In contrast, the transformation of ortho-substituted benzoates was poorly coupled to O(2) utilization, with >10 times more O(2) being consumed than benzoate. However, the apparent K(m) of TADO for these benzoates was >100 μM, indicating that they do not effectively inhibit the turnover of good substrates.