Reverse Ordered Sequential Mechanism for Lactoperoxidase with Inhibition by Hydrogen Peroxide

Lactoperoxidase (LPO, FeIII in its resting state in the absence of substrates)—an enzyme secreted from human mammary, salivary, and other mucosal glands—catalyzes the oxidation of thiocyanate (SCN−) by hydrogen peroxide (H2O2) to produce hypothiocyanite (OSCN−), which functions as an antimicrobial agent. The accepted catalytic mechanism, called the halogen cycle, comprises a two-electron oxidation of LPO by H2O2 to produce oxoiron(IV) radicals, followed by O-atom transfer to SCN−. However, the mechanism does not explain biphasic kinetics and inhibition by H2O2 at low concentration of reducing substrate, conditions that may be biologically relevant. We propose an ordered sequential mechanism in which the order of substrate binding is reversed, first SCN− and then H2O2. The sequence of substrate binding that is described by the halogen cycle mechanism is actually inhibitory.

Electrochemistry of single droplets of inverse (water-in-oil) emulsions

Single water droplet electrochemistry investigated for the first time, reveals the biphasic kinetics of ion transfer within water-in-oil emulsions.

The oligomeric state of thyroid receptor regulates hormone binding kinetics

We previously reported that mutations in the thyroid hormone receptor (TR) surface that mediates dimer and heterodimer formation do not alter affinity for cognate hormone (triiodothyronine (T3)) yet dramatically enhance T3 association and dissociation rates. This study aimed to show that TR oligomeric state influences binding and dissociation kinetics. We performed binding assays using marked hormone (125I-T3) and TRs expressed and purified by different methods. We find that T3 associates with TRs with biphasic kinetics in solution; a rapid step (half-life ±0.1 h) followed by a slower second step (half-life ±5 h) and that purification of monomers suggests that biphasic kinetics are due to the presence of monomers and dimers in our preparations. In support of this idea, incubation of TR ligand binding domain monomers with corepressor peptide induces dimer formation and decreases association rates and T3 binds to, and dissociates from, a TRβ mutant that only forms dimers (TRβD355R) with slow single-phase kinetics. In addition, heterodimer formation with retinoid X receptors also influences ligand binding kinetics. Together, these results suggest that the dimer/heterodimer surface is allosterically coupled to the hormone binding pocket and that different interactions at this surface exert different effects on ligand binding that may be relevant for TR actions in the cell.