The mechanism of amine oxidation catalyzed by D-arginine dehydrogenase (DADH) has been investigated using steady-state and rapid reaction kinetics, with pH, substrate and solvent deuterium kinetic isotope effects (KIE) as mechanistic probes, and computational studies. Previous results showed that 85-90% of the flavin reduction reaction occurs in the mixing time of the stopped-flow spectrophotometer when arginine is the substrate, precluding a mechanistic investigation. Consequently, leucine, with slower kinetics, has been used here as the flavin-reducing substrate. Free energy calculations and the pH profile of the K(d) are consistent with the enzyme preferentially binding the zwitterionic form of the substrate. Isomerization of the Michaelis complex, yielding an enzyme-substrate complex competent for flavin reduction, is established due to an inverse hyperbolic dependence of k(cat)/K(m) on solvent viscosity. Amine deprotonation triggers the oxidation reaction, with cleavage of the substrate NH and CH bonds occurring in an asynchronous fashion, as suggested by the multiple deuterium KIE on the rate constant for flavin reduction (k(red)). A pK(a) of 9.6 signifies the ionization of a group that facilitates flavin reduction in the unprotonated form. The previously reported high-resolution crystal structures of the iminoarginine and iminohistidine complexes of DADH allow us to propose that Tyr(53), on a mobile loop covering the active site, may participate in substrate binding and facilitate flavin reduction.