Kinetic mechanism of putrescine oxidase from Rhodococcus erythropolis.

Abstract

Putrescine oxidase from Rhodococcus 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.

DOI: 10.1111/febs.12945

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Cite this paper

@article{Kopacz2014KineticMO, title={Kinetic mechanism of putrescine oxidase from Rhodococcus erythropolis.}, author={Malgorzata M Kopacz and Dominic P. H. M. Heuts and Marco W Fraaije}, journal={The FEBS journal}, year={2014}, volume={281 19}, pages={4384-93} }