Quantifying the Dynamics of L-Kynureninase Orthologs during Catalysis Using HDX-MS.

The breakdown of tryptophan in eukaryotes and some bacteria can proceed via a L-Kynurenine (Kyn) intermediate. This intermediate has three potential fates; it can be converted to kynurenic acid, hydroxylated to 3-hydroxyl-kynurenine (3OH-Kyn), or hydrolyzed to L-anthranillic acid and alanine. L-Kynureninase (Kynase) is the aminotransferase enzyme responsible for hydrolysis of either Kyn or 3OH-Kyn. Kynase functions as a homodimer, with one chain contributing an essential pyridoxal-5-phosphate cofactor, and the other chain contributing three catalytic residues. High-resolution structural studies show that while human and bacterial Kynase orthologs are structurally similar, they have distinct catalytic residues. Enzyme studies also identify different substrate specificities, with the human ortholog preferring 3OH-Kyn and the bacterial ortholog preferring Kyn. The basis for this preference is beyond the catalytic residues, as simply switching active sites between orthologs fails to switch substrate preference. The goal of this study was to characterize the dynamics of Kynase enzymes during catalysis. To do this, we have used hydrogen/deuterium exchange coupled to mass spectrometry. Through a substantial series of experiments, we have compared the dynamics of several Kynase orthologs and engineered Kynase mutants. Data sets were collected for enzymes without substrate, as well as with Kyn and 3OH-Kyn. The results identify the determinants of substrate specificity and provide insight into mechanisms of catalysis. This information is invaluable in the design of Kynase-based enzyme therapeutics, as well as our general understanding of enzyme dynamics.