Inactivation of C30A trimethylamine dehydrogenase by N-cyclopropyl-alpha-methylbenzylamine, 1-phenylcyclopropylamine, and phenylhydrazine.
Trimethylamine dehydrogenase (TMADH) from the bacterium Methylophilus methylotrophus (sp. W(3)A(1)) and its C30A mutant were inactivated with three known inactivators of monoamine oxidase, namely, phenylhydrazine, N-cyclopropyl-alpha-methylbenzylamine, and 1-phenylcyclopropylamine. All three compounds irreversibly inactivated both the wild-type and C30A mutant enzymes, although phenylhydrazine was 10 times more potent than N-cyclopropyl-alpha-methylbenzylamine, which was much more potent than 1-phenylcyclopropylamine. The change in the UV--visible absorption spectra upon modification indicated that the flavin was modified. In the case of the C30A mutant, the absence of a covalent attachment of the flavin to the polypeptide has permitted LC-electrospray mass spectrometry of the reaction product to be undertaken, demonstrating new mass peaks corresponding to various chemically modified forms of the flavin cofactor. In the case of N-cyclopropyl-alpha-methylbenzylamine, masses corresponding to hydroxy-FMN and hydroxyriboflavin were detected. 1-Phenylcyclopropylamine inactivation of the C30A mutant produced three modified flavins, as evidenced by the electrospray mass spectrum: hydroxy-FMN, FMN plus C(6)H(5)COCH(2)CH(2), and hydroxy-FMN plus C(6)H(5)COCH(2)CH(2). Phenylhydrazine inactivation of the C30A mutant gave at least seven different modified flavins: hydroxyriboflavin, hydroxy-FMN, two apparently isomeric compounds corresponding to hydroxy-FMN plus one phenyl group, two apparently isomeric compounds corresponding to FMN plus one phenyl group, and FMN plus two phenyl groups. Covalent flavin adduct formation appears to be the only modification because dialysis of the inactive enzyme followed by reconstitution with FMN restores the enzyme activity to that of a noninactivated control.
Mitchell
D J
DJ
Department of Chemistry, Northwestern University, Evanston, Illinois 60208-3113, USA.
Nikolic
D
D
Jang
M H
MH
van Breemen
R B
RB
Hille
R
R
Silverman
R B
RB
D000409:Alanine / Q000235:genetics; D001596:Benzylamines / Q000494:pharmacology*; D002853:Chromatography, Liquid; D003521:Cyclopropanes / Q000494:pharmacology*; D003545:Cysteine / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005486:Flavin Mononucleotide / Q000737:chemistry; D008996:Monoamine Oxidase Inhibitors / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics*; D010659:Phenylhydrazines / Q000494:pharmacology*; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet
D001596:Benzylamines; D003521:Cyclopropanes; D004791:Enzyme Inhibitors; D008996:Monoamine Oxidase Inhibitors; D010659:Phenylhydrazines; C030299:phenylhydrazine; C032446:N-(1-methyl)cyclopropylbenzylamine; C045293:1-phenylcyclopropylamine; D005486:Flavin Mononucleotide; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase; D003545:Cysteine; D000409:Alanine