In mammalian cells, the flavoprotein spermine oxidase (SMO) catalyzes the oxidation of spermine to spermidine and 3-aminopropanal. Mechanistic studies have been conducted with the recombinant human enzyme. The initial velocity pattern in which the ratio between the concentrations of spermine and oxygen is kept constant establishes the steady-state kinetic pattern as ping-pong. Reduction of SMO by spermine in the absence of oxygen is biphasic. The rate constant for the rapid phase varies with the substrate concentration, with a limiting value (k3) of 49 s-1 and an apparent K d value of 48 μMat pH8.3. The rate constant for the slow step is independent of the spermine concentration, with a value of 5.5 s-1, comparable to the kcat value of 6.6 s-1. The kinetics of the oxidative half-reaction depend on the aging time after the spermine and enzyme are mixed in a double-mixing experiment. At an aging time of 6 s, the reaction is monophasic with a second-order rate constant of 4.2 mM-1 s-1. At an aging time of 0.3 s, the reaction is biphasic with two second-order constants equal to 4.0 and 40 mM-1 s-1. Neither is equal to the kcat/KO2 value of 13 mM -1 s-1. These results establish the existence of more than one pathway for the reaction of the reduced flavin intermediate with oxygen. The kcat/KM value for spermine exhibits a bell-shaped pH profile, with an average pKa value of 8.3. This profile is consistent with the active form of spermine having three charged nitrogens. The pH profile for k3 shows a pKa value of 7.4 for a group that must be unprotonated. The pKi-pH profiles for the competitive inhibitors N,N′-dibenzylbutane-1,4-diamine and spermidine show that the fully protonated forms of the inhibitors and the unprotonated form of an amino acid residue with a pKa of ∼7.4 in the active site are preferred for binding.
ASJC Scopus subject areas