To gain a deeper understanding of the reaction mechanism and speciation, we conducted a study on the amination of 13 C-2 using 13 C NMR spectroscopy. In a reaction that proceeds to 75% conversion after 50 h (Figure ), we observed excellent mass balance in the conversion of C-2 to 13 C-3 (purple line). Importantly, the major C-L-Lys-derived species (red) contained a nitrogen with an 15 N chemical shift of 293.9 ppm, which is characteristic of an imine species. This detection was achieved using a 1 H- N HMBC experiment at natural abundance, and correlations from the methylene 1 H resonances at 2.26 ppm and 2.19 ppm to the imine nitrogen were observed (see Supporting Information for details), consistent with the formation of the 3,4,5,6-tetrahydropyridine isomer C-1. This cyclic byproduct results from the spontaneous cyclization of its purported linear counterpart, 6-amino-2-oxohexanoic acid, which, according to our mechanistic hypothesis, plays a significant role in driving the equilibrium toward the formation of 3. Other unidentified species are also observed during the reaction that contribute to the slight decrease in mass balance between C-1 and 13 C-L-Lys (orange line, see Supporting Information for details). enzyme is saturated. Interestingly, both L-Lys and L-Orn elicit a lower KM than L-Ala (78 mM, 81 mM, and 563 mM, respectively), suggesting that the former two are better suited for amine transfer by PRO-TRANS(248) under practical reaction conditions, although L-Ala does indeed elicit faster initial reaction rates at concentrations exceeding ~200-300 mM. Mindful of the fact that KM is dependent on both the substrate and the enzyme, and thus subject to change in the context of a different transaminase, we anticipate that this analysis will prove nonetheless insightful when contemplating initial reaction conditions for future endeavors involving these smart donors.