Isotope effects suggest a stepwise mechanism for berberine bridge enzyme.
Biochemistry
; 51(37): 7342-7, 2012 Sep 18.
Article
em En
| MEDLINE
| ID: mdl-22931234
ABSTRACT
The flavoprotein Berberine Bridge Enzyme (BBE) catalyzes the regioselective oxidative cyclization of (S)-reticuline to (S)-scoulerine in an alkaloid biosynthetic pathway. A series of solvent and substrate deuterium kinetic isotope effect studies were conducted to discriminate between a concerted mechanism, in which deprotonation of the substrate phenol occurs before or during the transfer of a hydride from the substrate to the flavin cofactor and substrate cyclization, and a stepwise mechanism, in which hydride transfer results in the formation of a methylene iminium ion intermediate that is subsequently cyclized. The substrate deuterium isotope effect of 3.5 on k(red), the rate constant for flavin reduction, is pH-independent, indicating that C-H bond cleavage is rate-limiting during flavin reduction. Solvent isotope effects on k(red) are equal to 1 for both wild-type BBE and the E417Q mutant, indicating that solvent exchangeable protons are not in flight during or before flavin reduction, thus eliminating a fully concerted mechanism as a possibility for catalysis by BBE. An intermediate was not detected by rapid chemical quench or continuous-flow mass spectrometry experiments, indicating that it must be short-lived.
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Base de dados:
MEDLINE
Assunto principal:
Oxirredutases N-Desmetilantes
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Proteínas de Plantas
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Alcaloides de Berberina
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Cannabis
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Benzilisoquinolinas
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article