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 k_cat and K_DHA of 2.4 s⁻¹ and 0.7 μM, respectively, but no significant dependence on the concentration of O₂. A low-level inhibition (K_I = 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.

虽然酶 2,4'-二羟基苯乙酮双加氧酶 (DAD) 已为人所知数十年，但对其报道的底物 2,4'-二羟基苯乙酮 (DHA) 的 DAD 催化氧化裂解的机制知之甚少. 本研究的目的是确定活性金属中心并表征反应动力学的底物依赖性，为更深入的机理研究奠定基础。对此，DAD V1M 突变体（bDAD) 被过表达、纯化并用各种金属离子重组。进行了评估重构酶活性以及反应动力学的底物和产物依赖性的动力学测定。用各种金属离子重构载脂蛋白的结果支持酶活性需要 Fe ³⁺中心。反应速率显示 DHA 的简单饱和动力学，k _cat和K _DHA的值分别为2.4 s ^-1和 0.7 μM，但对 O ₂的浓度没有显着依赖性。低水平抑制（K _I = 1100 μM) 由 4HB 产物观察到。结果支持最小动力学模型，其中 DHA 与静止的三价铁酶结合，然后快速添加 O ₂以产生不可逆地塌陷为产物的中间复合物。