The enzymatic mechanism of sulphite formation in Saccharomyces cerevisiae was investigated using a purified 3'-phosphoadenylsulphate (PAPS) reductase and thioredoxin. The functionally active protein (MR 80-85 k) is represented by a dimer which reduces 3'-phosphoadenylyl sulphate to adenosine-3',5'-bisphosphate and free sulphite at a stoichiometry of 1:1. Reduced thioredoxin is required as cosubstrate. Examination of the reaction products showed that free anionic sulphite is formed with no evidence for "bound-sulphite(s)" as intermediate. Vmax of the enriched enzyme was 4-7 nmol sulphite.min-1.mg-1 using the homologous thioredoxin from yeast. The velocity of reaction decreased to 0.4 nmol sulphite.min-1.mg-1 when heterologous thioredoxin (from Escherichia coli) was used instead. The Km of homologous thioredoxin was 0.6.10(-6) M, for the heterologous cosubstrate it increased to 1.4.10(-6) M. The affinity for PAPS remained practically unaffected (Km PAPS: 19.10(-6) M in the homologous, and 21.10(-6) M in the heterologous system). From the kinetic data it is concluded that the enzyme followed an ordered mechanism with thioredoxin as first substrate followed by PAPS as the second. Parallel lines in the reciprocal and a common intersect in the Hanes-plots for thioredoxin were seen as indication of a ping-pong (with respect to thioredoxin) uni-bi (with respect to PAPS) mechanism.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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