Protein Engineering of Enzymes
| Project/Area Number |
06454654
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Functional biochemistry
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| Research Institution | Osaka University |
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Principal Investigator |
KURAMITU Seiki Osaka Univ., Fac.Sci., Professor, 理学部, 教授 (60153368)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥7,000,000 (Direct Cost: ¥7,000,000)
Fiscal Year 1995: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1994: ¥5,600,000 (Direct Cost: ¥5,600,000)
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| Keywords | Enzymatic Mechanism / One Enzyme-Two Substrates / Hydrophobic Substrate / Hydrophobic Interaction / Dynamics / Fluctuation / Protein Engineering / Gene Engineering / 一酵素二基質 / 多機能酵素 / 触媒反応機構 / 基質認識機構 / 部位特異的変異法 / キメラ / X線結晶解析 / アミノ基転移酵素 |
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| Research Abstract |
Many enzymes have reactivities for only one kind of substrate and their substrate specificities are very narrow. It was shown that the hydrophobic substrate specificity of aminotransferase is surprisingly broad. Aminotransferase catalyzes reversible transamination between amino acid and keto acid substratevia the ping-pong bi-bi mechanism. A series of aliphatic amino acids and keto acids with straight and terminal-branched side chains were used to study the hydrophobic substrate specificity. These substrates were useful for studying the effect of one additional substrate methyl group. The activity of aminotransferase generally increased with an increase in the chain length of the substrate. In particular, when the number of carbons of the substrate was between 4 and 7, the additional effect of one methyl group for stabilization of the transition state was constant. This suggests that the hydrophobic substrate pocket has a uniform hydrophobic environment and can equally recognize the methyl group in the substrate-binding pocket. It is suggested that the determinant of the hydrophobic substrate specificity is a spatial distribution of residues with plasticity in the substrate-binding pocket. The recognition of manifold hydrophobic substrates with different shapes requires precise re-packing by the enzyme and might, therefore, require flexibility of the binding pocket and/or the whole enzyme molecule. In other words, the strict substrate specificity is in contrast to the plasticity of the enzyme.This concept will be applicable to many enzymes that recognize hydrophobic substrates and will be useful for constructing a hydrophobic ligand binding protein.
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Report
(3 results)
Research Products
(44 results)
