caa a22 4500 397558007 CHVBK 20180308164808.0 cr unu---uuuuu 161202e199610 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021476 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021476 Characterization of Rat Monoamine Oxidase A with Noncovalently-Bound FAD Expressed in Yeast Cells [Elektronische Daten] [Iwao Hiro, Yukio Tsugeno, Isamu Hirashiki, Fusahiro Ogata, Akio Ito] 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 kynurainine. 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 noneovalently 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. © 1996 BY THE JOURNAL OF BIOCHEMISTRY Regular Papers nationallicence covalently-bound flavin nationallicence flavoprotein nationallicence monoamine oxidase A nationallicence pargyline nationallicence sitedirected mutagenesis nationallicence Hiro Iwao Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka 812-81 aut Tsugeno Yukio Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka 812-81 aut Hirashiki Isamu Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka 812-81 aut Ogata Fusahiro Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka 812-81 aut Ito Akio Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka 812-81 aut The Journal of Biochemistry Oxford University Press 120/4(1996-10), 759-765 0021-924X 120:4<759 1996 120 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021476 text/html Onlinezugriff via DOI 1 other jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021476 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Hiro Iwao Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka 812-81 aut NATIONALLICENCE 700 1- Tsugeno Yukio Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka 812-81 aut NATIONALLICENCE 700 1- Hirashiki Isamu Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka 812-81 aut NATIONALLICENCE 700 1- Ogata Fusahiro Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka 812-81 aut NATIONALLICENCE 700 1- Ito Akio Department of Chemistry, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka 812-81 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 120/4(1996-10), 759-765 0021-924X 120:4<759 1996 120 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford