| Project/Area Number |
07459023
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
広領域
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| Research Institution | Keio University |
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Principal Investigator |
OHTA Hiromichi Keio University, Department of Chemistry, Professor, 理工学部, 教授 (30152151)
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| Project Period (FY) |
1995 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1997: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1996: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1995: ¥4,000,000 (Direct Cost: ¥4,000,000)
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| Keywords | asymmetric decarboxylation / decarboxylase / enzyme / site-directed mutagenesis / substrate specificity / random mutation / 不斉脱炭素反応 / 活性部位特異的阻害剤 / 立体配座 / 活性化エントロビ- / CH-π相互作用 / チオールエステル結合 / システイン |
|---|
| Research Abstract |
Arylmalonate decarboxylase (AMDase) was isolated by us from a bacterium Alcaligenes bronchisepticus. It catalyzes asymmetric decarboxylation of disubstituted arylmalonates to give the optically active corresponding acetates. It is a very unique decarboxylation enzyme since it requires no coenzymes, such as biotin, coenzyme A,and ATP,which other decarboxylases and trancarboxylases do.
This enzyme consists of 240 amino acids, including four cysteine residues. Through the inhibition experiments, it was suggested that at least one of these cysteine residues is essential for the enzyme activity. Site-directed mutagenesis revealed that Cys188 is the one that is located in the active site.
Then, how does the Cys activate the substrates? How do the other amino acid residues in the binding site interact with the functional groups of the substrates? Physicochemical studies using well-designed inhibitors suggested that Cys residue forms a thiol ester bond with the substrates. Large electronegativity of thiol ester group is estimated to stabilize the transition state with a negative charge. Also kinetics of some specified substrates shed light on the conformation of the substrate in the active site. CH-pi interactions between enzyme and the substrate will be one of the binding forces. We challenged to widen the substrate specificity by random mutation. Although, we could not isolate a mutant of which substrate specificity had been widened, there were found a few mutants that were more active than the wild-type enzyme.
X-ray analysis for tertiary structure is now in progress.
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