Van Lanen, S.; Saryazdi, S.

Indolamine 2,3-dioxygenase (IDO1) is a hemoprotein that catalyzes the oxidative cleavage of L-tryptophan (L-Trp) to N'-formyl-L-kynurenine (L-NFK) along the kynurenine pathway. Its activity depletes L-Trp while initiating a signaling cascade culminating in an immunosuppressive outcome of clinical significance. The generally used in vitro activity assay for IDO1 relies on ascorbic acid and synthetic methylene blue, with the endogenous activator still uncertain. Here we demonstrate sodium hypochlorite, commonly known as bleach, functions as an in vitro activator/cofactor for the recombinant human IDO1-catalyzed dioxygenation reaction. Other hypohalous acids, generated in situ by lactoperoxidase (LPO) or in aqueous solutions of I2 or Br2, also activate IDO1. Contrastingly, the pseudohalide thoicyanate, a known, excellent substrate for LPO, yielded only trace levels of L-NFK. Importantly, complete conversion to L-NFK occurs with sub-stoichiometric hypohalous acid relative to L-Trp , and the overall reaction remains O2-dependent. Kinetic analysis with variable L-Trp and multiple, fixed concentrations of the activators/ cofactors revealed typical Michaelis-Menten kinetics without substrate inhibition, which contrasts past analysis using alternative IDO1 assays. The calculated second order rate constants were overall comparable regardless of the identity and concentration of hypohalous acid. Finally, 1-methyl-L-tryptophan, a reported inhibitor and poor substrate for rhIDO1, was reexamined with the hypohalous acid-dependent conditions revealing an improved catalytic efficiency when compared with the native substrate L-Trp. Along with this unanticipated result, the in vivo functional and mechanistic implications of the newly discovered hypohalous acid-dependent IDO1 activity are discussed.