1997 Fiscal Year Final Research Report Summary
Basic Studies on the Catalytic Reaction Mechanism of Enzymeby Organic Chemical Approaches
| Project/Area Number |
08456060
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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 |
Bioproduction chemistry/Bioorganic chemistry
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
ODA Jun'ichi Kyoto University, Inst.Chem.Res., Professor, 化学研究所, 教授 (50027041)
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| Co-Investigator(Kenkyū-buntansha) |
KATO Hiroaki Kyoto University, Inst.Chem.Res., Instructor, 化学研究所, 助手 (90204487)
HIRATAKE Jun Kyoto University, Inst.Chem.Res., Associate Professor, 化学研究所, 助教授 (80199075)
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| Project Period (FY) |
1996 – 1997
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| Keywords | organic syntesis / protein engineering / enzymatic reaction meehanism / Xray crystallography / transition-state analogue / glutathione / stereospecificity / molecular recognition |
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| Research Abstract |
In this study, we have investigated enzymatic reaction mechanism of ATP and NADPH dependent enzymes by X-ray crystallography, site-directed mutagenesis, and synthetic organie chemistry. We subjected three CN-bond ligases having different substrate specificity ; glutathione synthetase, gamma-glutamylcysteine synthetase, and asparagine synthetase. We also subjected two tropinone reductases with differnt stereospecificity ; tropinone reductase I and II.Each of these reductases only produce tropine or PSI-tropine that are diastereomeric with each other.
The following results were archived.
1.We succeed to crystallize gamma-glutamylcysteine synthetase by alteration of its surface cysteine residues into serine residues. We also synthesized its transition state analogue inhibitors. Kinetic analysis using the inhibitiors suggested some structural motives of the active site architecture of this enzme.
2.We crystallized two tropinone reductases and solved their three-dimensional structures by multiple isomorphous replacement methods independently. The results implicated the structural basis for their stereospecific reaction.
3.We determine theimportant active site residues of asparagine synthetase by site-directed mutageneses of those residues that proposec from crystal structure analysis. We also succeed to synthesize its transition-state analogue inhibitor and the crystal structure analysis of the enzyme complexed with the inhibitor is in progress.
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