Letter pubs.acs.org/JPCL
Substrate Inhibition in Human Indoleamine 2,3-Dioxygenase Benjamin Weber,† Elena Nickel,† Michael Horn,† Karin Nienhaus,† and G. Ulrich Nienhaus*,†,‡,§ †
Institute of Applied Physics (APH) and ‡Institute of Toxicology and Genetics (ITG), Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany § Department of Physics, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States S Supporting Information *
ABSTRACT: Human indoleamine 2,3-dioxygenase (hIDO) catalyzes the oxidative cleavage of the L-tryptophan (L-Trp) pyrrole ring. Catalysis is inhibited at high substrate concentrations; mechanistic details of this observation are, however, still under debate. Using time-resolved optical spectroscopy, we have analyzed the dynamics of ternary complex formation between hIDO, L-Trp, and a diatomic ligand. The physiological ligand dioxygen (O2) was replaced by carbon monoxide to exclude enzymatic turnover. Quantitative analysis of the kinetics reveals that the ternary complex forms whenever O2 binds first, whereas an LTrp substrate molecule arriving prior to O2 in the active site causes self-inhibition. Bound LTrp prevents the ligand from approaching the heme iron and, therefore, impedes formation of the catalytically active ternary complex.
SECTION: Kinetics and Dynamics
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ligand and substrate binding in hIDO using Fourier transform infrared (FTIR) spectroscopy.14 By using carbon monoxide (CO) as a sensitive internal electric field sensor that reports structural changes in its immediate vicinity,15−17 we showed that L-Trp in the active site acts as a roadblock for ligand migration to and from the binding site.18 We further noticed changes of the CO association kinetics at L-Trp concentrations above ∼5 mM, which could be due to the presence of L-Trp in a second site. However, there was no direct spectroscopic evidence supporting this interpretation. Recently, Efimov et al.11 suggested that self-inhibition can simply be accounted for by sequential binding of ligand and substrate to hIDO. To further elucidate self-inhibition of hIDO, we have revisited complex formation in ferrous hIDO by using flash photolysis at ambient temperature. Instead of the physiological ligand O2, we chose CO because it is not reactive toward L-Trp, so that we can focus on ligand and substrate dynamics in the absence of the ensuing enzymatic reaction. With this simplification, we have developed a kinetic model that quantitatively describes ternary complex formation (Figure 1A and B). Prior to the laser flash, the CO-saturated protein solution contains L-Trp free and L-Trp bound hIDO−CO in their equilibrium concentrations. The relative concentrations of these two species, denoted by hIDO−CO and hIDO−CO/W in the following, are governed by the equilibrium dissociation coefficient, Kd,W(CO) (Figure 1A). A nanosecond laser flash
nzymes catalyze specific metabolic reactions with high selectivity and can dramatically accelerate the conversion of a substrate to the final product. Exploring the mechanistic details of enzyme action at the molecular level is often a challenging endeavor. Structural studies are a prerequisite in this pursuit, but it is also essential to study protein dynamics and energetics to fully understand biomolecular reactions. Human indoleamine 2,3-dioxygenase (hIDO) is a heme enzyme that catalyzes the oxidative cleavage of the pyrrole ring of L-tryptophan (L-Trp, W) to produce N-formylkynurenine. In recent years, hIDO has emerged as an important therapeutic target for the treatment of cancer, chronic viral infections, and other diseases characterized by pathological immune suppression.1−5 For example, it has been noted that the expression of high levels of hIDO in tumors is directly correlated with a low survival rate.6−8 Therefore, blocking hIDO expression or inhibiting its activity may constitute effective strategies in cancer treatment.2 Substrate conversion of hIDO is initiated by formation of a ternary complex consisting of the enzyme, the diatomic ligand O2 coordinated to the ferrous heme iron, and the substrate, LTrp, bound in the active site. In the ensuing reaction, both oxygen atoms are inserted into the pyrrole ring. Interestingly, hIDO displays self-inhibition at high L-Trp concentrations for reasons that are still under debate.9−11 Initially, it was proposed that excess substrate binds directly to the ferric heme iron and, thereby, inhibits its reduction to the active ferrous state.12 More recently, however, it was suggested that substrate inhibition results from binding a second L-Trp molecule in an inhibitory site of the enzyme.9 In fact, a possible secondary substrate site close to the initial binding site was inferred from molecular dynamics simulations,13 which prompted us to investigate © 2014 American Chemical Society
Received: January 31, 2014 Accepted: February 6, 2014 Published: February 6, 2014 756
dx.doi.org/10.1021/jz500220k | J. Phys. Chem. Lett. 2014, 5, 756−761
The Journal of Physical Chemistry Letters
Letter
Figure 1. Mechanistic (A) and kinetic (B) schemes of gaseous ligand and substrate binding to hIDO−CO following CO photodissociation. Highlighted in gray: CO rebinding reaction. Figure 2. Flash photolysis kinetics of hIDO−CO dissolved in (A) 100 mM potassium phosphate buffer and (B,C) 75% glycerol/25% buffer (v/v) at 290 K. Samples were equilibrated with 1 atm of CO. Red: w/o L-Trp; green: 0.5 mM L-Trp; blue: 10 mM L-Trp. (C) Rebinding kinetics (in 75% glycerol/25% buffer) at different CO concentrations. Solid lines: 1 bar of CO; dashed lines: 0.33 bar. Short dashed and dotted lines: two concentrations
