title: 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.
authors: Molecular Enzymology Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
authors: Malgorzata M
authors: MM
authors: Kopacz
authors: Dominic P H M
authors: DP
authors: Heuts
authors: Marco W
authors: MW
authors: Fraaije
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D004138:Dinitrocresols / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry*; D011700:Putrescine / Q000737:chemistry; D012240:Rhodococcus / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D004138:Dinitrocresols; C024132:4,6-dinitro-o-cresol; C022488:putrescine oxidase; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011700:Putrescine
keywords: amine oxidase; flavin; kinetic mechanism; stopped-flow spectroscopy