Characterization of rat monoamine oxidase A with noncovalently-bound FAD expressed in yeast cells.
The FAD-binding cysteine of rat liver monoamine oxidase A (MAO A), Cys406, was converted to an alanine by site-directed mutagenesis of the cDNA. The wild-type and mutated enzymes were expressed in yeast cells and catalytic activities were assayed, using as substrates serotonin, tyramine, and kynuramine. Specific activities of the Ala-mutant for these substrates, calculated as the activities per pargyline-sensitive molecule, were about half of those of the wild-type enzyme. The Km values of the mutant enzyme for the substrates were similar to those of the wild-type enzyme. An adduct between FAD and pargyline, a mechanism-based inhibitor, was attached to the apoprotein in the wild-type enzyme, while in the Ala-mutant it was detached from the apoprotein, thereby indicating the presence of noncovalently bound FAD in the mutant enzyme. The Ala-mutant rapidly lost activity during incubation, whereas the wild-type enzyme retained the initial activity. Partial protection from inactivation occurred in the presence of FAD, but not of FMN. Recovery of the enzyme activity was nil when FAD was added after the inactivation. Thus, while the covalent attachment of FAD in MAO A is not required for the catalytic activity, it may function as a structural core for the active conformation in the membrane.
Hiro
I
I
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka.
Tsugeno
Y
Y
Hirashiki
I
I
Ogata
F
F
Ito
A
A
D000409:Alanine / Q000737:chemistry; D000818:Animals; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*; D007700:Kinetics; D007735:Kynuramine / Q000737:chemistry; D008099:Liver / Q000201:enzymology; D008995:Monoamine Oxidase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D008996:Monoamine Oxidase Inhibitors / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010293:Pargyline / Q000737:chemistry; D011485:Protein Binding; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012701:Serotonin / Q000737:chemistry; D013379:Substrate Specificity
D018076:DNA, Complementary; D008996:Monoamine Oxidase Inhibitors; D005182:Flavin-Adenine Dinucleotide; D012701:Serotonin; D007735:Kynuramine; D010293:Pargyline; D008995:Monoamine Oxidase; D003545:Cysteine; D000409:Alanine