1. Kinetic mechanism of putrescine oxidase from Rhodococcus erythropolis.
- Author
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Kopacz, Malgorzata M., Heuts, Dominic P. H. M., and Fraaije, Marco W.
- Subjects
PUTRESCINE ,RHODOCOCCUS erythropolis ,MONOAMINE oxidase ,DEAMINATION ,ENZYME kinetics ,FLAVIN adenine dinucleotide ,LIGAND binding (Biochemistry) - Abstract
Putrescine oxidase from Rhodococcus erythropolis (PuO) is a flavincontaining 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. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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