Document Detail

Simultaneous binding of coenzyme and two ligand molecules into the active site of fungal trihydroxynaphthalene reductase.
MedLine Citation:
PMID:  19071099     Owner:  NLM     Status:  MEDLINE    
We present here a kinetic characterization of the oxidation of the artificial substrate 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one in the presence of NADP(+) by trihydroxynaphthalene reductase from the filamentous fungus Curvularia lunata. Although the experimental data were gathered by conventional equipment and were only available for the reaction in one direction, the analysis confirms the bi-bi reaction mechanism and yields estimates of kinetic parameters of the intermediates. It is based on an independent estimation of coenzyme binding constants and on a sequential analysis of three portions of the progress curves, from the beginning of the reaction until equilibrium is reached. First, the plateaus are used to determine the overall equilibrium constant of the non-catalyzed reaction. Then, the dissociation constants of the oxidized and reduced cofactor are estimated by titration. Subsequently, the initial parts of the progress curves are analyzed using the rate equation that is derived under combined assumptions of equilibrium and steady state. The macroscopic relations obtained are then fixed in the final progress curve analysis where the information for only two remaining rate constants is extracted from their curved portions by fitting numerically solved model-specific differential equations to the data. At pH 8, the overall equilibrium largely favours the oxidized substrate and reduced cofactor, and the activity of the holoenzyme is inhibited by high substrate concentrations. Substrate inhibition can be discriminated from true cooperativity through the effects of apigenin, a flavonoid inhibitor that is structurally similar, but larger, than the substrate used in the study.
Jure Stojan; Mojca Brunskole; Tea Lanisnik Rizner
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Publication Detail:
Type:  Journal Article     Date:  2008-11-24
Journal Detail:
Title:  Chemico-biological interactions     Volume:  178     ISSN:  1872-7786     ISO Abbreviation:  Chem. Biol. Interact.     Publication Date:  2009 Mar 
Date Detail:
Created Date:  2009-02-09     Completed Date:  2009-02-27     Revised Date:  2012-05-25    
Medline Journal Info:
Nlm Unique ID:  0227276     Medline TA:  Chem Biol Interact     Country:  Ireland    
Other Details:
Languages:  eng     Pagination:  268-73     Citation Subset:  IM    
Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
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MeSH Terms
Ascomycota / enzymology*
Catalytic Domain
Coenzymes / metabolism*
Fungal Proteins / chemistry,  metabolism*
Oxidoreductases Acting on CH-CH Group Donors / chemistry,  metabolism*
Protein Binding
Reg. No./Substance:
0/Coenzymes; 0/Fungal Proteins; 0/Ligands; EC reductase; EC 1.3.-/Oxidoreductases Acting on CH-CH Group Donors

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