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 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 (1-PCPA) formed an irreversible flavin adduct whereas 2-phenylcyclopropylamine (2-PCPA) and N-cyclo-alpha-methylbenzylamine (N-CalphaMBA) 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-CalphaMBA 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.
Vintém
Ana Paula B
AP
Centre for Biomolecular Sciences, University of St. Andrews, St. Andrews, UK.
Price
Nigel T
NT
Silverman
Richard B
RB
Ramsay
Rona R
RR
D015632:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine / Q000378:metabolism; D000409:Alanine / Q000737:chemistry* / Q000235:genetics; D001596:Benzylamines / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D003521:Cyclopropanes / Q000737:chemistry / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D005415:Flavins / Q000378:metabolism; D006801:Humans; D007735:Kynuramine / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008995:Monoamine Oxidase / Q000737:chemistry* / Q000378:metabolism*; D008996:Monoamine Oxidase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D010084:Oxidation-Reduction; D010843:Pichia / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism
D001596:Benzylamines; D003521:Cyclopropanes; D005415:Flavins; D008996:Monoamine Oxidase Inhibitors; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D012701:Serotonin; D007735:Kynuramine; C067351:cyclopropylamine; D015632:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; D008995:Monoamine Oxidase; D003545:Cysteine; D000409:Alanine