MECHANISM OF INACTIVATION OF MONOAMINE OXIDASE-B BY THE ANTICONVULSANT AGENT MILACEMIDE (2-(N-PENTYLAMINO)ACETAMIDE)

The anticonvulsant agent milacemide (2-(n-pentylamino)acetamide) is kn own to inactivate monoamine oxidase-B (MAO-B). Various isotopically la beled analogues of milacemide are used to elucidate the mechanism of i nactivation of MAO-B by this compound. The metabolites of the oxidatio n of milacemide by MAO-B (pentanoic acid, pentanal, and glycinamide) a re shown not to be responsible for inactivation. MAO was inactivated w ith 2-(n-pentylamino)-acetamide (1a), 2-(n-pentylamino)[2,2-H-2(2)]ace tamide (1b), and 2-([1,1-H-2(2)]-n-pentylamino)acetamide(1c). Compound 1b exhibited little or no isotope effect on inactivation (k(inact)/K( I)) and 1c showed an isotope effect of 4.55 on k(inact)/K(I). These co mpounds also were found to be excellent substrates for MAO-B; lb showe d no isotope effect, but 1c exhibited an isotope effect of 4.53 on k(c at)/K(m). Incubation of MAO with 2-(n-pentylamino)[2-C-14] acetamide f ollowed by dialysis under denaturing conditions resulted in the incorp oration of 0.7 equiv of radioactivity per enzyme molecule. The same tr eatment with 2-([1-C-14]-n-pentylamino)acetamide led to the incorporat ion of 4 equiv of radioactivity into the enzyme. The excess radioactiv ity bound presumably arises from the [C-14]pentanal that is generated during turnover. In order to test this, MAO-B was incubated with [1-C- 14]pentylamine under similar conditions and 5.9 equiv of radioactivity was incorporated into the denatured enzyme. Therefore, the entire mol ecule becomes attached to the enzyme during inactivation. By following changes in the flavin absorption spectrum during inactivation with mi lacemide, it was shown that the flavin becomes reduced; however, denat uration of the inactivation enzyme causes flavin reoxidation under con ditions where radioactivity for 2-(n-pentylamino)[2-C-14]acetamide rem ains bound. This suggests that milacemide is oxidized during inactivat ion and the adduct results from attachment of milacemide to an amino a cid residue, not to the flavin cofactor. Inactivation with 2-(11-C-14] -n-pentylamino)acetamide produced [C-14]pentanoic acid and [C-14]-pent ylamine in the ratio of 92:8. Inactivation of MAO with 2-(n-pentylamin o)[2-C-14]acetamide gave [C-14]glycinamide and [C-14]oxamic acid, furt her supporting oxidation reactions at both the pentyl side chain and t he acetamido methylene. All of these results indicate that milacemide is oxidized at both the pentyl methylene and the acetamido methylene. Pentyl oxidation leads to inactivation, but it is not clear if acetami do methylene oxidation also leads to inactivation (Scheme I).