A lysine conserved in the monoamine oxidase family is involved in oxidation of the reduced flavin in mouse polyamine oxidase.
Lysine 315 of mouse polyamine amine oxidase corresponds to a lysine residue that is conserved in the flavoprotein amine oxidases of the monoamine oxidase structural family. In several structures, this lysine residue forms a hydrogen bond to a water molecule that is hydrogen-bonded to the flavin N(5). Mutation of Lys315 in polyamine oxidase to methionine was previously shown to have no effect on the kinetics of the reductive half-reaction of the enzyme (M. Henderson Pozzi, V. Gawandi, P.F. Fitzpatrick, Biochemistry 48 (2009) 1508-1516). In contrast, the mutation does affect steps in the oxidative half-reaction. The k(cat) value is unaffected by the mutation; this kinetic parameter likely reflects product release. At pH 10, the k(cat)/K(m) value for oxygen is 25-fold lower in the mutant enzyme. The k(cat)/K(O2) value is pH-dependent for the wild-type enzyme, decreasing below a pK(a) of 7.0, while this kinetic parameter for the mutant enzyme is pH-independent. This is consistent with the neutral form of Lys315 being required for more rapid flavin oxidation. The solvent isotope effect on the k(cat)/K(O2) value increases from 1.4 in the wild-type enzyme to 1.9 in the mutant protein, and the solvent inventory changes from linear to bowed. The effects of the mutation can be explained by the lysine orienting the bridging water so that it can accept the proton from the flavin N(5) during flavin oxidation. In the mutant enzyme the lysine amine would be replaced by a water chain.
Henderson Pozzi
Michelle
M
Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843-2128, USA.
Fitzpatrick
Paul F
PF
D000818:Animals; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D008995:Monoamine Oxidase / Q000737:chemistry* / Q000235:genetics; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000235:genetics; D000097667:Polyamine Oxidase
D008239:Lysine; D008995:Monoamine Oxidase; D000587:Oxidoreductases Acting on CH-NH Group Donors; D000097667:Polyamine Oxidase