Identification of reaction products and intermediates of aromatic-amine dehydrogenase by ¹⁵N and ¹³C NMR

¹³C- and ¹⁵N-NMR studies of the reaction of aromatic amine dehydrogenase (AADH) with methylamine demonstrated that the products of the reductive half-reaction are an equivalent of formaldehyde hydrate and a reduced aminoquinol form of the tryptophan tryptophylquinone (TTQ) cofactor which contains covalently bound substrate-derived N. These data are consistent with the Ping Pong kinetic mechanism and aminotransferase-type chemical reaction mechanism which have been previously proposed for AADH. Comparison of the ¹⁵N-NMR spectra of the aminoquinol TTQ intermediates of AADH and methylamine dehydrogenase (MADH) revealed that the substrate-derived aminoquinol N of AADH and MADH exhibited distinct ¹⁵N chemical shifts which are separated by approx. 7 p.p.m. In each case, the signal for the substrate-derived aminoquinol N appears optimally with short pulse delay and exhibits a relaxation time and chemical shift which are consistent with ¹⁵N covalently bound to an aromatic ring (i.e. aminoquinol) which is attached to a rigid protein matrix. The aminoquinol of AADH is less stable against reoxidation than that of MADH. These data suggest that differences in the active-site mediated electrostatic environments of the aminoquinol N in the respective enzymes may influence both the observed ¹⁵N chemical shift and the relative reactivities of the TTQ aminoquinols towards oxygen. These data also demonstrate the utility of ¹³C- and ¹⁵N-NMR spectroscopy as a tool for monitoring the intermediates and products of enzyme-catalysed transformations.