Carbinolamine reaction with AADH New insights into the reductive half-reaction mechanism of aromatic amine dehydrogenase revealed by reaction with carbinolamine substrates

Aromatic amine dehydrogenase uses a tryptophan tryptophylquinone (TTQ) cofactor to oxidatively deaminate primary aromatic amines. In the reductive half-reaction, a proton is transferred from the substrate C1 to Asp128β O2, in a reaction that proceeds by H-tunneling. Using solution studies, kinetic crystallography and computational simulation we show that the mechanism of oxidation of aromatic carbinolamines is similar to amine oxidation, but that carbinolamine oxidation occurs at a substantially reduced rate. This has enabled us to determine for the first time the structure of the intermediate prior to the H-transfer/reduction step. The proton–Asp128β O2 distance is ~3.7 Å, in contrast to the distance of ~2.7 Å predicted for the intermediate formed with the corresponding primary amine substrate. This difference of ~1.0 Å is due to an unexpected conformation of the substrate moiety, which is supported by MD-simulations and reflected in the ~10-fold slower TTQ reduction rate with phenylaminoethanol compared to that with primary amines. A water molecule is observed near TTQ C6 and is likely derived from the collapse of the preceding carbinolamine TTQ-adduct. We suggest this water molecule is involved in consecutive proton transfers following TTQ reduction, and is ultimately repositioned near the TTQ O7 concomitant with protein rearrangement. For all carbinolamines tested, highly stable amide-TTQ adducts are formed following proton abstraction and TTQ reduction. Slow hydrolysis of the amide occurs after, rather than prior to, TTQ oxidation and leads ultimately to a carboxylic acid product. The new structural data are consistent with the wide range of kinetic parameters observed with different amine substrates.