Catalytic activity of human indoleamine 2,3-dioxygenase (hIDO1) at low oxygen

Abstract

A cytokine-inducible extrahepatic human indoleamine 2,3-dioxygenase (hIDO1) catalyzes the first step of the kynurenine pathway. Immunosuppressive activity of hIDO1 in tumor cells weakens host T-cell immunity, contributing to the progression of cancer. Here we report on enzyme kinetics and catalytic mechanism of hIDO1, studied at varied levels of dioxygen (O2) and L-tryptophan (L-Trp). Using a cytochrome b5-based activating system, we measured the initial rates of O2 decay with a Clark-type oxygen electrode at physiologically-relevant levels of both substrates. Kinetics was also studied in the presence of two substrate analogs: 1-methyl-L-tryptophan and norharmane. Quantitative analysis supports a steady-state rather than a rapid equilibrium kinetic mechanism, where the rates of individual pathways, leading to a ternary complex, are significantly different, and the overall rate of catalysis depends on contributions of both routes. One path, where O2 binds to ferrous hIDO1 first, is faster than the second route, which starts with the binding of L-Trp. However, L-Trp complexation with free ferrous hIDO1 is more rapid than that of O2. As the level of L-Trp increases, the slower route becomes a significant contributor to the overall rate, resulting in observed substrate inhibition.

Indoleamine 2,3‐dioxygenase 1 (IDO1): an up‐to‐date overview of an eclectic immunoregulatory enzyme

“…Recent crystallographic studies of human IDO1 in complex with the substrate cast a light on the binding modes of L-Trp into the catalytic cleft and an accessory site, with the latter being associated to the inhibition by substrate phenomenon [3,20]. Upon oxygen binding to the ferrous active form of IDO1 [21][22][23][24], L-Trp binds to the catalytic pocket on the sixth coordination site of the heme cofactor, yielding the catalytically active ternary complex (Figure 3). According to this binding mode, the indole ring engages S167 with a water-mediated hydrogen bond, and establishes face-to-edge -stacking and hydrophobic interactions with F163 and Y126.…”

Advances in indoleamine 2,3-dioxygenase 1 medicinal chemistry

“…However, the interpretation of kinetic studies of inhibitors suffer from the complexity of the catalytic mechanism of the enzyme which follows a redox activation cycle, and a steady-state kinetic model being composed of two substrates ( l -Trp and oxygen) and two substrate recognition routes which lead to ternary complex formation. 39 Specifically, the redox activation cycle of the enzyme consists of a shift from a ferric inactive form to a ferrous catalytically active form of the heme group. This may affect inhibition kinetic studies of uncompetitive compounds showing similar binding affinity towards both ferric and ferrous forms of IDO1, and non-competitive compounds displaying preferential binding affinity towards the inactive ferric form of the enzyme.…”

“…Although both routes contribute to the overall rate of catalysis, increasing the concentration of l -Trp ( 1 ) favors the contribution of the second route and eventually leads to substrate inhibition. 39 …”

Different Mechanisms of Catalytic Complex Formation in Two L-Tryptophan Processing Dioxygenases

“…In this case, the enzymatic activity is rate-limited by L-Trp dissociation. Of note, self-inhibition may well be incomplete at physiologically relevant L-Trp concentrations if there is a small probability that ligands can bypass the bulky substrate (Kolawole et al, 2015 ).…”