The role of halogen substituents and substrate pKa in defining the substrate specificity of 2,6-dichlorohydroquinone 1,2-dioxygenase (PcpA)
JBIC Journal of Biological Inorganic Chemistry
Abstract2,6-Dichlorohydroquinone 1,2-dioxygenase (PcpA) is a non-heme Fe(II) enzyme that is speci... more Abstract2,6-Dichlorohydroquinone 1,2-dioxygenase (PcpA) is a non-heme Fe(II) enzyme that is specific for ortho-dihalohydroquinones. Here we deconvolute the role of halogen polarizability vs. substrate pKa in defining this specificity, and show how substrate binding compares to the structurally homologous catechol extradiol dioxygenases. The substrates 2,6-dichloro- and 2,6-dibromohydroquinone (polarizable halogens, pKa1 = 7.3), 2,6-difluorohydroquinone (nonpolarizable halogens, pKa1 = 7.5), and 2-chloro-6-methylhydroquinone (polarizable halogen, pKa1 = 9.0) were examined through spectrophotometric titrations and steady-state kinetics. The results show that binding of the substrates to the enzyme decreased $$pK_{a1}^{\text{app}}$$pKa1app by about 0.5, except for 2,6-difluorohydroquinone, which showed no change. Additionally, the Kd values of 2,6-dichloro- and 2,6-dibromohydroquinone are about equal to their respective $$K_{\text{mA}}^{\text{app}}$$KmAapp. For comparison, with catechol 2,3-dioxygenase (XylE), the substrates 4-methyl- and 3-bromocatechol are bound to the enzyme exclusively in the monoanion form over a wide pH range, indicating a $$\Delta pK_{a1}^{\text{app}}$$ΔpKa1app of at least − 2.9 and − 1.2, respectively. The steady-state kinetic studies showed that 2,6-difluorohydroquinone is a poor substrate, with $${{k_{\text{cat}}^{\text{app}} } \mathord{\left/ {\vphantom {{k_{\text{cat}}^{\text{app}} } {K_{\text{mA}}^{\text{app}} }}} \right. \kern-0pt} {K_{\text{mA}}^{\text{app}} }}$$kcatapp/KmAapp approximately 40-fold lower and $$K_{\text{mA}}^{\text{app}}$$KmAapp 20-fold higher than 2,6-dichlorohydroquinone, despite its similar pKa1. Likewise, the pH dependence of $$k_{\text{cat}}^{\text{app}}$$kcatapp for 2-chloro-6-methylhydroquinone is nearly identical to that of 2,6-dichlorohydroquinone, despite its very different pKa1. These results show that (1) it is clearly the halogen polarizability and not the lower substrate pKa that determines the substrate specificity of PcpA, and (2) that PcpA, unlike the catechol extradiol dioxygenases, lacks an active site base that assists with substrate deprotonation, highlighting a key functional difference in what are otherwise similar active sites that defines their different reactivity.
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