Phosphoenolpyruvate- and ATP-dependent dihydroxyacetone kinases: covalent substrate-binding and kinetic mechanism.
Dihydroxyacetone (Dha) kinases are a sequence-conserved family of enzymes, which utilize two different phosphoryldonors, ATP in animals, plants, and some bacteria, and a multiphosphoprotein of the phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) in most bacteria. Here, we compare the...
| Main Authors: | , , , , , , , |
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| Format: | Journal article |
| Language: | English |
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2004
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| _version_ | 1797065145525993472 |
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| author | Garcia-Alles, L Siebold, C Nyffeler, T Flükiger-Brühwiler, K Schneider, P Bürgi, H Baumann, U Erni, B |
| author_facet | Garcia-Alles, L Siebold, C Nyffeler, T Flükiger-Brühwiler, K Schneider, P Bürgi, H Baumann, U Erni, B |
| author_sort | Garcia-Alles, L |
| collection | OXFORD |
| description | Dihydroxyacetone (Dha) kinases are a sequence-conserved family of enzymes, which utilize two different phosphoryldonors, ATP in animals, plants, and some bacteria, and a multiphosphoprotein of the phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) in most bacteria. Here, we compare the PTS-dependent kinase of Escherichia coli and the ATP-dependent kinase of Citrobacter freundii. They display 30% sequence identity. The binding constants of the E. coli kinase for eleven short-chain carbonyl compounds were determined by acetone precipitation of the enzyme-substrate complexes. They are 3.4 microM for Dha, 780 microM for Dha-phosphate (DhaP), 50 microM for D,L-glyceraldehyde (GA), and 90 microM for D,L-glyceraldehyde-3-phosphate. The k(cat) for Dha of the PTS-dependent kinase is 290 min(-1), and that of the ATP-dependent kinase is 1050 min(-1). The Km for Dha of both kinases is <6 microM. The X-ray structures of the enzyme-GA and the enzyme-DhaP complex show that substrates as well as products are bound in hemiaminal linkage to an active-site histidine. Quantum-mechanical calculations offer no indication for activation of the reacting hydroxyl group by the formation of the hemiaminal. However, the formation of the hemiaminal bond allows selection for short-chain carbonyl compounds and discrimination against structurally similar polyols. The Dha kinase remains fully active in the presence of 2 M glycerol, and phosphorylates trace impurities of carbonyl compounds present in glycerol. |
| first_indexed | 2024-03-06T21:24:28Z |
| format | Journal article |
| id | oxford-uuid:429a6f68-0e67-4ef7-b323-c4a87eee984a |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T21:24:28Z |
| publishDate | 2004 |
| record_format | dspace |
| spelling | oxford-uuid:429a6f68-0e67-4ef7-b323-c4a87eee984a2022-03-26T14:50:29ZPhosphoenolpyruvate- and ATP-dependent dihydroxyacetone kinases: covalent substrate-binding and kinetic mechanism.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:429a6f68-0e67-4ef7-b323-c4a87eee984aEnglishSymplectic Elements at Oxford2004Garcia-Alles, LSiebold, CNyffeler, TFlükiger-Brühwiler, KSchneider, PBürgi, HBaumann, UErni, BDihydroxyacetone (Dha) kinases are a sequence-conserved family of enzymes, which utilize two different phosphoryldonors, ATP in animals, plants, and some bacteria, and a multiphosphoprotein of the phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) in most bacteria. Here, we compare the PTS-dependent kinase of Escherichia coli and the ATP-dependent kinase of Citrobacter freundii. They display 30% sequence identity. The binding constants of the E. coli kinase for eleven short-chain carbonyl compounds were determined by acetone precipitation of the enzyme-substrate complexes. They are 3.4 microM for Dha, 780 microM for Dha-phosphate (DhaP), 50 microM for D,L-glyceraldehyde (GA), and 90 microM for D,L-glyceraldehyde-3-phosphate. The k(cat) for Dha of the PTS-dependent kinase is 290 min(-1), and that of the ATP-dependent kinase is 1050 min(-1). The Km for Dha of both kinases is <6 microM. The X-ray structures of the enzyme-GA and the enzyme-DhaP complex show that substrates as well as products are bound in hemiaminal linkage to an active-site histidine. Quantum-mechanical calculations offer no indication for activation of the reacting hydroxyl group by the formation of the hemiaminal. However, the formation of the hemiaminal bond allows selection for short-chain carbonyl compounds and discrimination against structurally similar polyols. The Dha kinase remains fully active in the presence of 2 M glycerol, and phosphorylates trace impurities of carbonyl compounds present in glycerol. |
| spellingShingle | Garcia-Alles, L Siebold, C Nyffeler, T Flükiger-Brühwiler, K Schneider, P Bürgi, H Baumann, U Erni, B Phosphoenolpyruvate- and ATP-dependent dihydroxyacetone kinases: covalent substrate-binding and kinetic mechanism. |
| title | Phosphoenolpyruvate- and ATP-dependent dihydroxyacetone kinases: covalent substrate-binding and kinetic mechanism. |
| title_full | Phosphoenolpyruvate- and ATP-dependent dihydroxyacetone kinases: covalent substrate-binding and kinetic mechanism. |
| title_fullStr | Phosphoenolpyruvate- and ATP-dependent dihydroxyacetone kinases: covalent substrate-binding and kinetic mechanism. |
| title_full_unstemmed | Phosphoenolpyruvate- and ATP-dependent dihydroxyacetone kinases: covalent substrate-binding and kinetic mechanism. |
| title_short | Phosphoenolpyruvate- and ATP-dependent dihydroxyacetone kinases: covalent substrate-binding and kinetic mechanism. |
| title_sort | phosphoenolpyruvate and atp dependent dihydroxyacetone kinases covalent substrate binding and kinetic mechanism |
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