1997 Fiscal Year Final Research Report Summary
Photoaffinity Labeling Reagents for Thermostable Diphosphate Synthase
| Project/Area Number |
08680626
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bioorganic chemistry
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| Research Institution | Yamagata University |
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Principal Investigator |
MAKI Yuji Yamagata University, Department of Material and Biological Chemistry, Professor, 理学部, 教授 (00007171)
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| Project Period (FY) |
1996 – 1997
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| Keywords | farnesyl diphosphate synthase / substrate analog / photolabeling regent / thermostable enzyme / geranyl diphosphate / dimethylallyl diphosphate / isopentenyl diphosphate / hexaprenyl diphosphate |
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| Research Abstract |
In this study, two types of photoactivatable analogs of isoprenyl diphosphates have been synthesized. One type is geranyl diphosphate analogs having arylazide group as photoactivatable group and other type is isoprenyl diphosphate analogs which contain photoacitivatable benzoylphenoxy group. The analogs of both types have been evaluated as substrates and inhibitors of isoprenoid synthases including thermostable farnesyl diphosphate synthase (FPS) from Bacillus stearothermophilus.. The radiolabeled analogs (substituted at the hydrogen of C-1 of analogs with ^3H). of both types were also synthesized.
The geranyl diphosphate analogs containing rho-lazido aryl group has been shown to be competetive inhibitors (Ki =10.5 muM). When farnesyl diphosphate synthase (FPS) with the inhibitors was irradiated by UV-lamp, the complete loss of enzyme activity was observed. On the other hand, no loss of activity was observed in the absence of the inhibitor. The protein photolabeled with the radiolabeled inhibitor was collected and subjected on SDS-PAGE,insicating that the radioactive protein was produced and the band on PAGE appeared at the almost same position to that of wild FPS.
The DMAPP and GPP analogs containing benzophenone as photoactivatable group were evalued as substrate and inhibitors three bacterial prenyl transferases : FPS,hexaprenyl diphosphate synthase (HexPS) and undecaprenyl diphoshate synthase (UPS). These analogs undergo chain extension and will allow identification of hydrophobic active site residues of these enzymes.
In conclusion, aryl azide analog studied here is useful for FPS as probing tools and the analogs having benzoylphenoxy are useful for the prenyl transferases which produce longer prenols.
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