pH dependence of a mammalian polyamine oxidase: insights into substrate specificity and the role of lysine 315.
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.
Henderson Pozzi
Michelle
M
Departments of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.
Gawandi
Vijay
V
Fitzpatrick
Paul F
PF
D000818:Animals; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D051379:Mice; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D000097667:Polyamine Oxidase
D008239:Lysine; D000587:Oxidoreductases Acting on CH-NH Group Donors; D000097667:Polyamine Oxidase