The metal- and substrate-dependences of 2,4′-dihydroxyacetophenone dioxygenase

Abstract

While the enzyme, 2,4′-dihydroxyacetophenone dioxygenase (DAD), has been known for decades, very little has been characterized of the mechanism of the DAD-catalyzed oxidative cleavage of its reported substrate, 2,4′-dihydroxyacetophenone (DHA). The purpose of this study was to identify the active metal center and to characterize the substrate-dependence of the kinetics of the reaction to lay the foundation for deeper mechanistic investigation. To this, the DAD V1M mutant (bDAD) was overexpressed, purified, and reconstituted with various metal ions. Kinetic assays evaluating the activity of the reconstituted enzyme as well as the substrate- and product-dependences of the reaction kinetics were performed. The results from reconstitution of the apoprotein with a variety of metal ions support the requirement for an Fe3+ center for enzyme activity. Reaction rates showed simple saturation kinetics for DHA with values for kcat and KDHA of 2.4 s−1 and 0.7 μM, respectively, but no significant dependence on the concentration of O2. A low-level inhibition (KI = 1100 μM) by the 4HB product was observed. The results support a minimal kinetic model wherein DHA binds to resting ferric enzyme followed by rapid addition of O2 to yield an intermediate complex that irreversibly collapses to products.