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 eysteine 374 was mutated to alanine and the purified enzyme characterized kinetically. The mutant was active but had decreased k(cat)/K-m 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 (I-PCPA) formed an irreversible flavin adduct whereas 2-phenylcyclopropylamine (2-PCPA) and N-cyclo-α-methylbenzylamine (N-Cα MBA) 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-CaMBA 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. © 2005 Elsevier Ltd. All rights reserved.