Site-directed mutagenesis of monoamine oxidase A and B: role of cysteines.
Nine cysteines are found in the deduced amino acid sequences of both human liver monoamine oxidase (MAO)-A and MAO-B. The role of these cysteine residues in MAO-A and -B catalytic activity was studied by site-directed mutagenesis, whereby each cysteine residue was converted to serine. The wild-type and mutant cDNAs were then transiently transfected into COS cells and assayed for MAO-A and -B catalytic activity using 5-[3H]hydroxytryptamine and [14C]phenylethylamine, respectively, as substrates. Catalytic activities were retained in seven MAO-A cysteine to serine mutants (mutations at residues 165, 210, 266, 306, 321, 323, and 398) and in six MAO-B cysteine to serine mutants (mutations at residues 5, 172, 192, 297, 312, and 389). Kinetic parameters (Km) of these mutants were also similar to those of the wild-type enzymes, indicating that these cysteines are not necessary for enzymatic activity. Substitution of MAO-A Cys-374 and -406 and MAO-B Cys-156, -365, and -397 with serine resulted in complete loss of MAO-A and -B catalytic activity. The loss of catalytic activity was not due to unsuccessful transfection of the mutants, as indicated by either Northern blot or Western blot analysis. The loss of catalytic activity in the MAO-A Ser-406 and MAO-B Ser-397 mutants may be due to the prevention of covalent binding of the enzyme to the cofactor FAD, which is necessary for catalytic activity. The loss of catalytic activity of MAO-A Ser-374 and MAO-B Ser-156 and -365 suggests that these cysteines are important for catalytic activity, but whether they are involved in forming the active site or are important for the appropriate conformation of MAO-A and -B remains to be studied.
Wu
H F
HF
Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles 90033.
Chen
K
K
Shih
J C
JC
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry*; D004247:DNA / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D008995:Monoamine Oxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D014162:Transfection
D004247:DNA; D008995:Monoamine Oxidase; D003545:Cysteine