2019 Fiscal Year Annual Research Report
Determining sulfur isotope fractionation values of individual enzymes and how they evolve through time.
| Project/Area Number |
18H01325
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| Research Institution | Tokyo Institute of Technology |
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Principal Investigator |
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| Co-Investigator(Kenkyū-buntansha) |
上野 雄一郎 東京工業大学, 理学院, 教授 (90422542)
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| Project Period (FY) |
2018-04-01 – 2021-03-31
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| Keywords | enzyme mechanism / sequence evolution / kinetic isotope effect / sulfate reduction / geobiology |
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| Outline of Annual Research Achievements |
In this year, we discovered that the kinetic parameters of the Aps reductase enzyme are modulated by the product. We now determined the enzyme kinetics under non-inhibited conditions using an assay which specifically removes the inhibitor from the assay solution. The Km, and Vmax properties of the enzyme are dramatically altered, which in turn affects predicted whole cell sulfur isotope fractionation. We are now analyzing this effect in a whole cell model which relates sulfate reduction rate, thermodynamics, and enzyme kinetic isotope fractionation factors.. Whether or not the altered kinetics of the enzyme affect the apparent KIE of the enzyme is under investigation. Furthermore, we have now demonstrated active enzyme from heterologous expression, and are now in the position to assay the sulfur isotopes of that enzyme from multiple bacterial sources, including deltaproteobacteria, firmicutes, chloroflexi, and members of the archaea. In addition to these enzyme focussed experiments, we have also conducted work using resting cells, and demonstrated an effect of cell respiratory power to the standing pool of ATP/ADP. Altogether, we will be able to use our results to understand whole cell isotope fractionation of sulfur from the enzyme, and evolutionary perspectives.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
First of all, we have demonstrated the importance of the Aps enzyme in sulfur isotope fractionation. Second, we have discovered product inhibition in the enzyme, which revises our understanding of the overall pathway kinetics. Finally, we demonstrated heterologous expression can produce active enzyme. In the final year of the project we will be able to measure the isotopes from these enzymes.
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| Strategy for Future Research Activity |
The plan for our future work remains the same as in the initial proposal. We now have active enzyme in the laboratory, and when it is re-opened from the COVID virus problem, we will pursue our goals.
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Research Products
(5 results)
