Human FAD synthase (isoform 2): a component of the machinery that delivers FAD to apo-flavoproteins
| Autor: | Teresa Anna Giancaspero, Michele Galluccio, Cesare Indiveri, Maria Barile, Stefania Iametti, Elisabetta Gianazza, Enza Maria Torchetti, Francesco Bonomi |
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| Rok vydání: | 2011 |
| Předmět: |
chemistry.chemical_classification
ATP synthase biology Flavoprotein Cell Biology Plasma protein binding Biochemistry enzymes and coenzymes (carbohydrates) Chaotropic agent Enzyme chemistry Catalytic cycle biology.protein bacteria heterocyclic compounds Binding site FMN adenylyltransferase Molecular Biology |
| Zdroj: | FEBS Journal. 278:4434-4449 |
| ISSN: | 1742-464X |
| DOI: | 10.1111/j.1742-4658.2011.08368.x |
| Popis: | A soluble form of human FAD synthase (isoform 2; hFADS2) was produced and purified to homogeneity as a recombinant His-tagged protein. The enzyme binds 1 mole of the FAD product very tightly, although noncovalently. Complete release of FAD from the 'as isolated' protein requires extensive denaturation. A 75 : 25 mixture of apo/holoprotein could be prepared by treatment with mild chaotropes, allowing estimatation of the contribution made by bound FAD to the protein stability and evaluatation of whether structural rearrangements may be required for FAD release. Under turnover conditions, the enzyme catalyzes FAD assembly from ATP and FMN and, at a much lower rate, the pyrophosphorolytic hydrolysis of FAD. Several mechanistic features of both reactions were investigated in detail, along with their dependence on environmental conditions (pH, temperature, dependence on metals). Our data indicate that FAD release may represent the rate-limiting step of the whole catalytic cycle and that the process leading to FAD synthesis, and delivery to client apoproteins may be tightly controlled. |
| Databáze: | OpenAIRE |
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