Change of lactate dehydrogenase function by protein engineering
| Project/Area Number |
08660120
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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 |
応用微生物学・応用生物化学
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| Research Institution | Science University of Tokyo |
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Principal Investigator |
TAGUCHI Hayao Science University of Tokyo, Department of Applied Bioscience Associate professor, 理工学部, 助教授 (90188136)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1997: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1996: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | lactate dehydrogenase / catalytic center / substrate specificity / stereochemistry / allosteric enzyme / enzyme regulation / 3-D protein structure / change of enzyme function / アロステリック |
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| Research Abstract |
ln Lactobocillus pentosus D-LDH,replacements of Phe299 induced marked reductions in the affinity to 2-ketoacid substrates and the catalytic velocity, and also changed primary isotope effects on the enzyme reaction, indicating that Phe299 plays essential roles to stimulate the binding of 2-ketoacid substrates and subsequent catalytic reaction. The replacements Particularly markedly damaged the pyruvate reduction among the reactions for 2-ketoacids, indicating that Phe299 plays also important role in the specific recognition of the substrate C3 moiety. Replacement of Serl02 induced a significant positive cooperativity on the substrate binding, indicating that the structure of the substrate binding site is coordinately changed with the whole protein structure. In L.casei allosteric L-LDH,on the other hand, replacement of His205 in the regulatory site induced a great loss of the homotropic regulation by pyruvate, and thereby change the enzyme to a absolutely fructose 1,6-bisphosphate-dependent enzyme. Analysis by the enzyme kinetics and chemical modification strongly suggested that His205 is involved in the pyruvate binding not in an inactive state, but in an active state of the enzyme structure. Therefore, His205 possibly triggered the allosteric transition through the interactions with pyruvate. In L.pentosus non-allosteric L-LDH,which shows high sequence similarity with L.casei L-LDH,the three-dimensional structure could be built through its crystarographic analysis. To also determine three dimensional structure of L.casei L-LDH,in addition, the crystals of the enzyme, the maximal size of which was I.25 mm, were obtained by screening optimal conditions for crystallization.
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Report
(3 results)
Research Products
(6 results)
