The Mechanism-based Inactivation of 2,3-Dihydroxybiphenyl 1,2-Dioxygenase by Catecholic

2,3-Dihydroxybiphenyl 1,2-dioxygenase (EC 1.13.11.39), the extradiol dioxygenase of the biphenyl biodegradation pathway, is subject to inactivation during the steadystate cleavage of catechols. Detailed analysis revealed that this inactivation was similar to the O2-dependent inactivation of the enzyme in the absence of catecholic substrate, resulting in oxidation of the active site Fe(II) to Fe(III). Interestingly, the catecholic substrate not only increased the reactivity of the enzyme with O2 to promote ring cleavage but also increased the rate of O2-dependent inactivation. Thus, in air-saturated buffer, the apparent rate constant of inactivation of the free enzyme was (0.7 0.1) 10 3 s 1 versus (3.7 0.4) 10 3 s 1 for 2,3-dihydroxybiphenyl, the preferred catecholic substrate of the enzyme, and (501 19) 10 3 s 1 for 3-chlorocatechol, a potent inactivator of 2,3-dihydroxybiphenyl 1,2-dioxygenase (partition coefficient 8 2, Km app 4.8 0.7 M). The 2,3-dihydroxybiphenyl 1,2-dioxygenase-catalyzed cleavage of 3-chlorocatechol yielded predominantly 2-pyrone6-carboxylic acid and 2-hydroxymuconic acid, consistent with the transient formation of an acyl chloride. However, the enzyme was not covalently modified by this acyl chloride in vitro or in vivo. The study suggests a general mechanism for the inactivation of extradiol dioxygenases during catalytic turnover involving the dissociation of superoxide from the enzyme-catecholic-dioxygen ternary complex and is consistent with the catalytic mechanism.