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Kinetic mechanism of putrescine oxidase from R hodococcus erythropolis
Author(s) -
Kopacz Malgorzata M.,
Heuts Dominic P. H. M.,
Fraaije Marco W.
Publication year - 2014
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12945
Subject(s) - flavin group , chemistry , oxidative deamination , ternary complex , reaction rate constant , imine , stereochemistry , photochemistry , cofactor , substrate (aquarium) , hydride , enzyme kinetics , electron transfer , medicinal chemistry , catalysis , enzyme , active site , kinetics , organic chemistry , hydrogen , physics , oceanography , quantum mechanics , geology
Putrescine oxidase from R hodococcus erythropolis (PuO) is a flavin‐containing amine oxidase from the monoamine oxidase family that performs oxidative deamination of aliphatic diamines. In this study we report pre‐steady‐state kinetic analyses of the enzyme with the use of single‐ and double‐mixing stopped‐flow spectroscopy and putrescine as a substrate. During the fast and irreversible reductive half‐reaction no radical intermediates were observed, suggesting a direct hydride transfer from the substrate to the FAD . The rate constant of flavin reoxidation depends on the ligand binding; when the imine product was bound to the enzyme the rate constant was higher than with free enzyme species. Similar results were obtained with product‐mimicking ligands and this indicates that a ternary complex is formed during catalysis. The obtained kinetic data were used together with steady‐state rate equations derived for ping‐pong, ordered sequential and bifurcated mechanisms to explore which mechanism is operative. The integrated analysis revealed that PuO employs a bifurcated mechanism due to comparable rate constants of product release from the reduced enzyme and reoxidation of the reduced enzyme–product complex.

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