1-Phenylcyclopropylamine (1-PCPA) is shown to be a mechanism-based inactivator of mitochondrial monoamine oxidase (MAO). The strained cyclopropyl ring is important to inactivation since α,α-dimethylbenzylamine, the acyclic analogue of 1-PCPA, is neither an inactivator nor a substrate of MAO. Two different pathways occur during inactivation by 1-PCPA, both believed to be derived from a common intermediate. One pathway leads to irreversible inactivation of the enzyme and a 1:1 stoichiometry of radioactivity to the active site when l-[phenyl-14C]PCPA is used as the inactivator; the other pathway results in a covalent reversible adduct. Three organic reactions are carried out on the irreversibly labeled enzyme in order to determine the structure of the active site adduct. Sodium boro[3H]hydride reduction results in the incorporation of 0.73 equiv of tritium, suggesting a carbonyl functionality. Baeyer-Villiger oxidation followed by saponification gives 0.8 equiv of phenol, indicating the presence of a phenyl ketone. Treatment of the labeled enzyme with hydroxide produces acrylophenone, as would be expected from the retro-Michael reaction of β-X-propiophenone. The identity of X is determined in two ways. The optical spectrum of the flavin cofactor is reduced during inactivation; no reoxidation occurs upon denaturation. Pronase treatment of the radioactively labeled enzyme produces fragments that contain both the radioactivity and the flavin. The X group, therefore, is the flavin. The results of two tests designed to differentiate N5 from C4a attachment to the flavin suggest an N5 adduct. In addition to formation of this stable covalent adduct, another pathway occurs 7 times as often. This alternate reaction of l-[phenyl-14C]PCPA with MAO produces 7 equiv of [14C] acrylophenone during the course of irreversible inactivation and is believed to arise from formation of the same type of adduct as described above except that X is something other than the N5-flavin (Y). Upon denaturation of this labeled enzyme, the flavin is completely oxidized when most of the radioactivity is still bound to the enzyme. This indicates that Y is not a C4a-flavin adduct and suggests attachment to an active site amino acid residue. More facile elimination of Y from this β-substituted propiophenone adduct would give acrylophenone on the time scale of the inactivation. Treatment of the reversible adduct with sodium borohydride prior to denaturation prevents release of radioactivity. This is further evidence for the involvement of a retro-Michael reaction in the formation of acrylophenone. The rate constant for the reversible reactivation component was determined to be 7.25 times larger than that for the irreversible inactivation component, and this is consistent with the observed 7:1 ratio for these reactions. Suggestions are made as to how and why attachment to the amino acid residue occurs. The mechanism that is consistent with these results is a one-electron transfer from 1-PCPA to the flavin followed by homolytic cyclopropyl ring opening to the common radical intermediate, which is trapped by two different active site radicals. Flavin radical combination yields the stable adduct; Y-capture produces the labile adduct.
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