pH dependence of a mammalian polyamine oxidase: insights into substrate specificity and the role of lysine 315

Abstract

Mammalian polyamine oxidases (PAOs) catalyze the oxidation of N1-acetylspermine and N1-acetylspermidine to produce N-acetyl-3-aminopropanaldehyde and spermidine or putrescine. Structurally, PAO is a member of the monoamine oxidase family of flavoproteins. The effects of pH on the kinetic parameters of mouse PAO have been determined to provide insight into the protonation state of the polyamine required for catalysis and the roles of ionizable residues in the active site in amine oxidation. For N1-acetylspermine, N1-acetylspermidine, and spermine, the k(cat)/K(amine)-pH profiles are bell-shaped. In each case, the profile agrees with that expected if the productive form of the substrate has a single positively charged nitrogen. The pK(i)-pH profiles for a series of polyamine analogues are most consistent with the nitrogen at the site of oxidation being neutral and one other nitrogen being positively charged in the reactive form of the substrate. With N1-acetylspermine as the substrate, the value of k(red), the limiting rate constant for flavin reduction, is pH-dependent, decreasing below a pK(a) value of 7.3, again consistent with the requirement for an uncharged nitrogen for substrate oxidation. Lys315 in PAO corresponds to a conserved active site residue found throughout the monoamine oxidase family. Mutation of Lys315 to methionine has no effect on the k(cat)/K(amine) profile for spermine; the k(red) value with N1-acetylspermine is only 1.8-fold lower in the mutant protein, and the pK(a) in the k(red)-pH profile with N1-acetylspermine shifts to 7.8. These results rule out Lys315 as a source of a pK(a) in the k(cat)/K(amine) or k(cat)/k(red) profiles. They also establish that this residue does not play a critical role in amine oxidation by PAO.

Figure 1

A, k_cat/K_amine-pH profile of wild type PAO with N1-acetylspermine (□), N1-acetylspermidine (●), and spermine (△), and K315M PAO (◆) with spermine. The lines are from fits of the data to eq 3. B, Theoretical protonation states of N1-acetylspermine with no proton, 1 proton, 2 protons, or 3 protons.

Figure 2

pK_i-pH profile of wild type PAO with (A) N1-acetyl-N3-pentyl-1,3-diaminopropane (●), 1,8-diaminooctane (□), and N1-acetyl-1,8-diaminooctane (▲) and (B) N1-acetyl-1,12-diaminododecane (■) and 1,12-diaminododecane (○). The lines for N1-acetyl-1,8-diaminooctane and N1-acetyl-1,12-diaminododecane are from fits to eq 1, for N1-acetyl-N3-pentyl-1,3-diaminopropane to eq 2, and for 1,8-diaminooctane and 1,12-diaminododecane to eq 3.

Figure 3

The reduction of PAO by 1 mM N1-acetylspermine at pH 7.5, 20 °C. (A) Absorbance changes at 458 nm upon reduction of 20 µM wild-type PAO by 1 mM N1-acetylspermine. The line is from a fit to eq 4. (B) Absorbance spectra of flavin intermediates observed in the reductive half reaction of wild-type PAO. (C) The dependence of the individual rate constants on the N1-acetylspermine concentration for wild-type (first phase(●), second phase (■) and third phase (◆)) and K315M (first phase (○) and second phase (□)) PAO. The lines are from fits of the concentration dependence of the rate constant for the first phase to the Michaelis-Menten equation. (D) Absorbance spectra of the flavin intermediates observed in the reductive half reaction of K315M PAO.

Figure 4

pH dependence of k_red for wild type (●) and K315M (□) PAO with N1-acetylspermine at 20 °C. The lines are from fits of the data to eq 2.

Figure 5

Relative positions of the conserved active site lysine and the FAD in human MAO A (blue carbons), human MAO B (purple carbons), maize PAO (yellow carbons), S. cerevisiae spermine oxidase Fms1 (green carbons), Calloselasma rhodostoma L-amino acid oxidase (gray carbons), and human LSD1 (light brown carbons). This figure was composed from PDB files 2BXS (MAO A), 1OJ9 (MAO B), 1H83 (maize PAO), 1RSG (Fms1), 1F8S (L-amino acid oxidase), and 2HKO (LSD1). To generate the figure, the atoms of the central pyrazine rings of the FAD cofactors were overlaid. The water molecule shown is from maize PAO.

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