The role of halogen substituents and substrate pKa in defining the substrate specificity of 2,6-dichlorohydroquinone 1,2-dioxygenase (PcpA).

2,6-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 [Formula: see text] 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 [Formula: see text]. 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 [Formula: see text] of at least - 2.9 and - 1.2, respectively. The steady-state kinetic studies showed that 2,6-difluorohydroquinone is a poor substrate, with [Formula: see text] approximately 40-fold lower and [Formula: see text] 20-fold higher than 2,6-dichlorohydroquinone, despite its similar pKa1. Likewise, the pH dependence of [Formula: see text] 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.

Development of a new statewide eating disorder service: The role of evidence in a real world setting.

OBJECTIVE: There are three aims of this report. First, to describe how research evidence informed a service development rationale for a new statewide eating disorder service (SEDS) for people aged 15 years and older. Second, to examine the profile of people accessing SEDS in the first 2 years of its operation with respect to the three broad dimensions: illness stage, illness severity, and previous history of treatment. Finally, to examine which patient characteristics resulted in the recommendation of ongoing treatment contact with SEDS. METHOD: Over a 2-year period (July 2014 to July 2016) 292 people were referred to the service, 171 (59%) who consented to have their data used in research. RESULTS: Half of the referrals related to anorexia nervosa (AN; 51.2%), with the remainder split between bulimia nervosa (25.3%) and other specified feeding and eating disorders (23.5%); 65.9% had previously received treatment for an eating disorder. The initial information about the service was typically provided by the general practitioner/primary care physician. Compared with any other eating disorder diagnosis, people with AN were three times more likely to be recommended to retain treatment contact with SEDS. DISCUSSION: Service development informed by research evidence, clinical expertise, and consideration of patients' characteristics, values, and circumstances, allows for a flexible but accountable development strategy.