Studies on the Role of Lysine‐296 in Human Mitochondrial Monoamine Oxidase B Catalysis

Abstract

Monoamine oxidase B (MAO B) is an outer membrane-bound mitochondrial flavoenzyme that catalyzes the oxidative deamination of biogenic amines and neurotransmitters with the reduction of O2 to H2O2. The production of H2O2by elevated levels of this enzyme on aging is thought to contribute to age-related neurodegenerative diseases. Recent structural data on human MAO B show the ε-amino group of Lys-296 is H-bonded to N(5) of the flavin ring via a water molecule; a structural motif also found in several other oxidase flavoenzymes. Recent theoretical calculations on MAO B catalysis suggest Lys-296 to play functional roles in the O2 reaction of reduced MAO B with O2to form H2O2in stabilizing the intermediate O2.− - flavin radical pair and in H+ donation in the second electron transfer step to form H2O2. To provide experimental support for a functional role of Lys-296 in MAO B catalysis, Lys-296 has been mutated to Arg and to Gln respectively and the mutant enzymes expressed in P. pastoris and characterized. Western Blot analyses using an antisera specific for covalent flavin show that MAO B K296R and MAO B K296Q each contain covalent FAD. Only the K296R mutant enzyme exhibits any detectable catalytic activity with benzylamine (BA) as substrate (kcat =5.2 min−1; ~1% of WT MAO B) and a higher level of activity with phenethylamine (PEA) (kcat =23.8 min−1; ~10% of WT MAO B). The reductive half reaction constitutes the rate-limiting step in catalysis with BA (Dkcat = 5.7) and with PEA(Dkcat = 4). WT MAO B exhibits respective Dkcat values (4.7 and 1.2) for these substrates. The O2reaction rate of MAO B K296R (as estimated from kcat/KmO2) is 6times slower than that observed with wild-type MAO B. These results are consistent with the calculations where the increased basicity of Arg relative to Lys results in a slower rate of the reduced enzyme reaction with O2. Supported by NIH Grant GM-29433