This award in the Chemistry of Life Processes (CLP) program supports work by Professors Denis Rousseau and Syun-Ru Yeh at the Albert Einstein College of Medicine to carry out fundamental studies on the energy landscape that underlies the catalytic mechanism of two enzyme systems: nitric oxide synthase and indolamine dioxygenase. This research will be done as part of an International Collaboration in Chemistry (ICC) with Professor Uli Nienhaus at the University of Karlsruhe in Germany, whose work will be supported by the Deutsche Forschungsgemeinschaft (DFG). Nitric oxide synthase catalyzes the formation of the signaling molecule, nitric oxide, from L-arginine and oxygen; and indolamine dioxygenase regulates immune responses by oxidizing L-tryptophan to N-formylkynurenine. To determine the dynamic properties that modulate the energy landscapes of the enzymatic reactions of these two proteins, critical amino acid residues will be mutated and their structural and catalytic properties will be the studied by spectroscopic methods under equilibrium and kinetic conditions. The mutants will be sent to the Nienhaus lab in Karlsruhe for the dynamics measurements by temperature derivative Fourier transform infrared spectroscopy; the data thus generated will be compared to the basic structural and functional properties obtained at the Albert Einstein College of Medicine. The process will be repeated until a comprehensive understanding of the role of dynamics in these two enzymes is achieved. In order to obtain a sound foundation of the catalytic mechanisms, the data will be integrated by combined quantum mechanics and molecular mechanics calculations.
The collaboration will include visits by both groups to each other's lab to set the strategy for the research and evaluate the findings. These visits between the US and German groups will provide outstanding training opportunities to all of the students and postdoctoral fellows participating in this project. The research will reveal how the molecular dynamics of each enzyme dictate its functional properties and will therefore serve as a foundation for the development of therapeutic targets for these enzymes.
This project focused on the determination of the fundamental properties of two important human enzymes: Indoleamine 2,3-Dioxygenase (IDO) and nitric oxide synthase. IDO catalyzes the oxidative cleavage of L-Tryptophan (L-Trp), the scarcest essential amino acid in mammals. The majority of our dietary L-Trp is metabolized in the liver by an analogous heme-based dioxygenase, Tryptophan Dioxygenase (TDO). In contrast to the hepatic TDO, IDO is ubiquitously distributed in all tissues other than liver and instead of regulating homeostatic serum Trp concentrations like TDO, IDO is inducible by interferon-γ and is important for immunomodulation by regulating the tryptophan catabolism in T cells. It thereby plays a critical in the anti-tumor and antimicrobial activities. Consequently, IDO has attracted a great deal of attention recently owing to its potential as a therapeutic target for cancer. We identified a binding site in the human enzyme that binds the substrate, tryptophan, and inhibits the catalytic activity. In addition, the studies carried out in this project confirmed and extended the understanding of a new sequential oxygen insertion mechanism discovered by us. In this mechanism molecular oxygen binds to the central heme group of the enzyme and then each of the two oxygen atoms insert into the Trp by independent distinct steps (Figure 1). Several spectroscopic and computational studies clarified this mechanism. This work has opened a new window for research on heme dioxygenases led by our group and followed up by others all over the world in order to define the specific molecular interactions in this two-step mechanism and how it is regulated by the amino acids that are near the heme. Nitric oxide synthase (NOS) is the enzyme that generates nitric oxide (NO), which plays a critical role in nearly every physiological process. NO is formed from oxygen and the amino acid arginine at a catalytic site comprised of a heme group. There are three isoforms of the enzyme in humans. One plays a role in memory, one in immunology and one in the regulation of blood flow. Despite their physiological importance, their catalytic mechanisms are not fully understood because the identification of the intermediates in catalytic processes has been elusive. In this project by mutating critical residues, the catalytic process was modulated so as to reveal heretofore undetected intermediates. This has allowed for a near complete understanding of the catalytic process. Furthermore, it was discovered that distortion of the heme group, which is central to the catalysis, can be induced by amino acid groups near it (Figure 2) and can in turn modify its function. This has general implications for the functional properties of many other heme proteins.
