Mechanistic studies of mouse polyamine oxidase with N1,N12-bisethylspermine as a substrate

Abstract

In mammalian cells, the flavoprotein polyamine oxidase catalyzes a key step in the catabolism of polyamines, the oxidation of N1-acetylspermine and N1-acetylspermidine to spermidine and putrescine, respectively. The mechanism of the mouse enzyme has been studied with N1,N12-bisethylspermine (BESPM) as a substrate. At pH 10, the pH optimum, the limiting rate of reduction of the flavin in the absence of oxygen is comparable to the k(cat) value for turnover, establishing reduction as rate-limiting. Oxidation of the reduced enzyme is a simple second-order reaction. No intermediates are seen in the reductive or oxidative half-reactions. The k(cat) value decreases below a pK(a) of 9.0. The k(cat)/K(m) value for BESPM exhibits a bell-shaped pH profile, with pK(a) values of 9.8 and 10.8. These pK(a) values are assigned to the substrate nitrogens. The rate constant for the reaction of the reduced enzyme with oxygen is not affected by a pH between 7.5 and 10. Active site residue Tyr430 is conserved in the homologous protein monoamine oxidase. Mutation of this residue to phenylalanine results in a 6-fold decrease in the k(cat) value and the k(cat)/K(m) value for oxygen due to a comparable decrease in the rate constant for flavin reduction. This moderate change is not consistent with this residue forming a tyrosyl radical during catalysis.

Scheme 1

Scheme 2

Figure 1

Reductive half-reaction of polyamine oxidase with BESPM as the substrate: (A) visible absorbance spectra after mixing polyamine oxidase with BESPM (1.2 mM final concentration) anaerobically at pH 10 and 30 °C and (B) time dependence of the absorbance change at 465 nm in panel A. The line in panel B is from a fit to eq 3.

Figure 2

Oxidative half-reaction of polyamine oxidase with BESPM as the substrate: (A) time dependence of the absorbance change at 465 nm after mixing reduced polyamine oxidase with oxygen (0.12 mM final concentration) at pH 10 and 30 °C and (B) dependence of the rate constant for flavin oxidation on oxygen concentration. The line in panel A is from a fit to eq 3.

Figure 3

pH dependence of polyamine oxidase with BESPM as the substrate: (A) pH–k_cat profile and (B) pH–k_cat/K_BESPM profile. T = 30 °C. The lines are from fits of the data to eq 1 (A) and eq 2 (B).