Mutation of surface cysteine 374 to alanine in monoamine oxidase A alters substrate turnover and inactivation by cyclopropylamines

Modification of cysteine (Cys) residues inactivates monoamine oxidases (MAO) yet the crystal structure shows no conserved cysteines in the active site of MAO A (Ma, J. et al. J. Mol. Biol.2004, 338, 103–114). MAO A cysteine 374 was mutated to alanine and the purified enzyme characterized kinetically. The mutant was active but had decreased kcat/Km values compared to the wild-type enzyme. Cyclopropylamine-containing mechanism-based inactivators similarly showed lower turnover rates. Spectral studies and measurement of free thiols established that 1-phenylcyclopropylamine (1-PCPA) formed an irreversible flavin adduct whereas 2-phenylcyclopropylamine (2-PCPA) and N-cyclo-α-methylbenzylamine (N-CαMBA) formed adducts that allowed reoxidation of the flavin on denaturation and decreased cysteine in both wild-type and mutant MAO A. In the 1-PCPA and N-CαMBA inactivations, the partition ratio was decreased by more than 50% in the mutant. The data suggest that mutation of Cys374 influences MAO A catalysis, which has implications for MAO susceptibility to redox damage. These results are compared with previous work on the equivalent residue in MAO B, namely, cysteine 365.

The Cys374Ala mutant of monoamine oxidase A has 30% less activity than the wild-type but N-cyclopropyl-α-methylbenzylamine (3) still modified the mutant enzyme. A revised inactivation mechanism proposes generation of an α,β-unsaturated iminium ion, which escapes the active site because of the lack of an appropriate nucleophile to react with it.Figure optionsDownload as PowerPoint slide