Quantitative Structure-Activity Relationship for the Cleavage of C3/C4-Substituted Catechols by a Prototypal Extradiol Catechol Dioxygenase with Broad Substrate Specificity

抄録

Catechol 2, 3-dioxygenase [EC 1. 13. 11. 2] from Pseudomonas putida mt-2 (Mpc) catalyzes the extradiol cleavage of catechol to produce 2-hydroxymuconate semialdehyde. The K_m values for the catecholic substrate (K_mA) and O₂ (K_mO2), and catalytic constants (k_cat) were kinetically determined for eight C3/C4-substituted catechols at 25°C and pH 6.5 or 7.5. The first pK_a values (pK₁) were determined for eleven catechols (pK₁=7.26-9.47), correlated with Hammett substituent constants, and electron-withdrawing substituents significantly stabilized the monoanionic species of free catechols. Mpc preferred catechols with non-ionic substituents at the C3 or C4 position. 3-Phenylcatechol, a biphenyl, was cleaved, while 4-tert-butylcatechol was not. The logarithm of k_cat/K_mA (substrate specificity constant) exhibited a good linear correlation with pK₁ with the exception of those for 4-halocatechols. The logarithm of k_cat/K_mO2 showed a good linear correlation with pK₁ with the exception of that of 3-phenylcatechol. These results demonstrate that catechol binding to the Mpc active site, the following O₂ binding, and the activation of the bound O₂ are all sensitive to electronic effects of the substituents. However, k_cat did not correlate significantly with pK₁. The present study distinguishes clearly between the electronic and the steric effects of catecholic substrates in the reactivity of Mpc, and provides important insight into the mechanistic basis for a vast range of substrate specificities of extradiol dioxygenases.