PcpA is a bacterial non-heme Fe(II) enzyme that oxidatively cleaves 2,6-dichlorohydroquinone as a part of the pentachlorophenol (PCP) degradation pathway of Sphingobium chlorophenolicum. It has been shown to be specific for ortho-dihalohydroquinones. Possible sources of this specificity include the substrate pKa, and halogen bonding and/or metal-halogen secondary bonding, both of which depend upon halogen polarizability. Substrate binding titrations showed a similar small shift in pKa values between the free substrate and the substrate bound to the enzyme for all substrates. This suggests that PcpA may lack an active site base needed to deprotonate the substrate, in contrast to the closely related catechol extradiol dioxygenase enzymes. Steady-state kinetic studies showed that 2,6-difluorohydroquinone is a poor substrate, similar to 2,6-methylhydroquinone, unlike 2,6-dichloro- and 2,6- dibromohydroquinone. The pH dependence of the kinetics of these substrates provides additional insights into the role of substrate pKa. Together, these studies suggest that both a polarizable halogen substituent and the pKa of the substrate play important roles in defining the substrate specificity of PcpA.
