2007 Fiscal Year Final Research Report Summary
Elucidation of the substrate recognition and catalytic mechanism of sphingomyelinase
| Project/Area Number |
18590084
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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 |
Biological pharmacy
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| Research Institution | Osaka University of Pharmaceutical Sciences |
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Principal Investigator |
FUJII Shinobu Osaka University of Pharmaceutical Sciences, 薬学部, Senior Assistant Professor (80218966)
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| Project Period (FY) |
2006 – 2007
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| Keywords | Sphinaomyelinase / Catalytic mechanism / Substrate recognition / Substrate analog / Enzyme Inhibition |
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| Research Abstract |
Sphingomyelinase (SMase) catalyzes the hydrolysis of sphingomyelin (SM) to yield ceramide. Previously, we reported that the carbon and nitrogen analogs of SM, in which the oxygen of the phosphate ester to be hydrolyzed by SMase had been replaced, inhibited the activity of Bacillus cereus SMase. In the present study, the difluoromethylene analog, in which the oxygen of the phosphate ester had been replaced, was synthesized and it inhibited the activity of SMase with nearly the same attitude as the nitrogen analog.
The catalytic activities of phospholipases have been known to be strongly dependent on the physical states of the lipid substrates (e.g. monodispersed or micellar). However, this phenomenon has not yet been well-studied for SMases, since water-soluble SM is not easily available. In the present study, water-soluble substrates (lyso-PCs having different acyl chain-lengths and thiophosphate analog of SM (thio-SM) having sulfur atom instead of phosphate oxygen) were used to examine the kinetics and substrate-interactions with B. cereus SMase. When the initial velocities were plotted against the concentrations of substrates (lyso-PC and thio-SM), a dramatic increase at one-third of the critical micellar concentrations (cmcs) of substrates and no significant increase at the cmcs were observed. Furthermore, in the presence of Smase, short-chain phosphatidylcholine (PC), which is not hydrolyzed by Smase, aggregated at the below concentration of cmc. The interaction between Smase and PC was studied using a surface plasmon resonance based biosensor. Three types of sensorgrams for PC binding onto the Smase-immobilized chip were obtained at the concentration range around the cmc. These results indicate that the Smase possesses the interfacial recognition site, and the monodispersed substrate molecules cooperatively aggregate at this site.
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Research Products
(16 results)
