Mechanistic studies of human spermine oxidase: kinetic mechanism and pH effects

Abstract

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 (k(3)) of 49 s(-1) and an apparent K(d) value of 48 microM at pH 8.3. The rate constant for the slow step is independent of the spermine concentration, with a value of 5.5 s(-1), comparable to the k(cat) 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 k(cat)/K(O(2)) 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 k(cat)/K(M) value for spermine exhibits a bell-shaped pH profile, with an average pK(a) value of 8.3. This profile is consistent with the active form of spermine having three charged nitrogens. The pH profile for k(3) shows a pK(a) value of 7.4 for a group that must be unprotonated. The pK(i)-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 pK(a) of approximately 7.4 in the active site are preferred for binding.

Figure 1

Double reciprocal plot of the initial rate vs. the spermine concentration at a fixed ratio of spermine to oxygen concentrations of two at pH 8.3, 25°C.

Figure 2

k_cat/K_M-pH profile for spermine oxidase with spermine as substrate. The line is from a fit of the data to eq 2.

Figure 3

pK_i-pH profiles for spermidine (circles) and DBDB (triangles) as inhibitors of spermine oxidase. The lines are from fits to eq 2.

Figure 4

Absorbance spectra of flavin intermediates observed in the reductive half reaction of spermine oxidase (16.8 µM) by spermine at pH 8.3: 1, spectrum of spermine oxidase before reaction; 2, spectrum at the end of the first phase; 3, final spectrum.

Figure 5

pH dependence of the rate constant for reduction of spermine oxidase by spermine at 25°C. The line is from a fit of the data to eq 3.

Figure 6

Absorbance changes during the reaction of reduced spermine oxidase with 607 µM oxygen at pH 8.3 after first allowing the enzyme to react with spermine for 6 s (1) or 0.3 s (2). For clarity, only every fifth point is shown. The lines are from fits of the data to eq 4 or 6.

Figure 7

The dependence of the rate constants for flavin oxidation on the oxygen concentration at pH 8.3, 25°C, when enzyme and spermine are first allowed to react for 6 s (A) or 0.3 s (B).

Figure 8

Comparison of the observed rates of spermine oxidation by spermine oxidase as a function of oxygen concentration (circles) with values calculated from the mechanism of Scheme 4 and the kinetic parameters in Table 3 (triangles), at pH 8.3, 25°C. The line is from a fit of the calculated data to the Michaelis-Menten equation.

Scheme 1

Scheme 2

Scheme 3

Scheme 4