Substrate-Protein Interaction in Human Tryptophan dioxygenase

Abstract

The initial and rate-limiting step of the kynurenine pathway involves the oxidation of L-Trp to N-formyl kynurenine catalyzed by two hemeproteins, Trypophan 2,3 dioxygeanse (hTDO) and indoleamine 2,3-dioxygeanse (hIDO). Although hTDO and hIDO catalyze the same reaction, and show high structural homology, they are engaged in distinct physiological functions and show different biochemical properties. IDO has been implicated in diverse range of pathophysiological conditions, whereas TDO deals with the systemic regulation of the Trp flux in our body. Hence, understanding the differences between hTDO and hIDO offer invaluable information for the design of new inhibitors selective for hIDO. We have expressed, purified and characterized hTDO for the first time and demonstrated that the distal pocket of the two heme enzymes are distinctly different (JACS. 2007, 129, 15690-15701). In hTDO, the distal H76 residue is believed to act as an active site base to deprotonate the indole NH group of L-Trp, the initial step of the L-Trp oxidation reaction. In hIDO, this histidine residue is replaced by a serine. To investigate the role of the H76 residue in hTDO, we have constructed two mutants, in which the H76 is replaced by a serine or an alanine, and studied their structural and functional properties. Resonance Raman studies indicate that L-Trp is positioned in the active site by the ammonium, the carboxylate and the indole groups, via intricate H-bonding and hydrophobic interactions. This scenario is consistent with the observation that L-Trp binding significantly perturbs the electronic properties of the O2-complex of hTDO. The electronic properties of the active ternary complex of hTDO are found to be sensitive to the mutation of the H76 residue, highlighting the critical role of H76 in modulating the oxygen chemistry of hTDO.