Analysis of Allosteric mechanism in bacterial L-lactate dehydrogenases

细菌L-乳酸脱氢酶的变构机制分析

• 批准号: 04454069
• 负责人: OHTA Takahisa
• 金额: $ 4.29万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04454069/
• 关键词: L-Lactate dehydrogenase; Allosteric; X-Ray crystallography; Protein engineering; Substrate inhibition

1. X-ray crystal analysis of mutant enzyme : Mutant enzymes in which arginine-173 or histidine-188 in FBP-binding site were replaced to glutamine or tyrosine were obtained. Preliminary analysis of the enzymes showed that the histidine residue is necessary for the binding of FBP.2. Analysis of substrate inhibition : Replace experiments showed that serine-193 and serine-318 play an important role for determining Michaelis and inhibition constants.3. X-ray analysis of crystal which comprises both T and R forms of the LDH : Crystal analysis revealed that there are enzyme molecules in both T and R states in a single crystal. Both molecular structure were analyzed in 2.5 angstrom resolution.4. Molecular mechanism of allosteric phenomena : Binding of FBP in T state overcomes electrostatic repulsion between subunits in P-axis. This induces rotation among four subunits and makes the active site active by interaction between subunit in Q-axis.

1.突变酶的X射线晶体分析：获得了其中FBP结合位点中的精氨酸-173或组氨酸-188被谷氨酰胺或酪氨酸取代的突变酶。对酶的初步分析表明，组氨酸残基是结合FBP所必需的。底物抑制分析：替代实验表明丝氨酸-193和丝氨酸-318对米氏常数和抑制常数的测定起重要作用.包含LDH的T和R形式的晶体的X射线分析：晶体分析显示在单晶中存在T和R状态的酶分子。在2.5埃分辨率下分析了两种分子结构.变构现象的分子机制：T状态的FBP结合克服了P轴亚基之间的静电排斥。这诱导了四个亚基之间的旋转，并通过Q轴上亚基之间的相互作用使活性位点活化。

项目成果

Iwata,So: "Molecular Basis of Allosteric Activation of Bacterial L-Lactate Dehydrogenase" J.Mol.Biol.230. 21-27 (1993)

Iwata，So：“细菌 L-乳酸脱氢酶变构激活的分子基础”J.Mol.Biol.230。

Iwata, So: "Two states in crystals of bacterial L-lactate dehydrogenase reveal the molecular mechanism for allosteric control" Nature Structural Biology. (in press). (1994)

Iwata，So：“细菌 L-乳酸脱氢酶晶体的两种状态揭示了变构控制的分子机制”《自然结构生物学》。

Iwata, So: "A Study on the Allosteric Transition of L-lactate Dehydrogenase by Protein Crystallography" Nihon Kesshogaku kaisi. 35. 14-20 (1993)

Iwata，So：“通过蛋白质晶体学研究 L-乳酸脱氢酶的变构转变”Nihon Kesshogaku kaisi。

Koide,Shohei: "Conformational Equilibrium of an Enzyme Catalytic Site in the Allosteric Transition" Biochemistry. 31. 5362-5368 (1992)

Koide,Shohei：“变构转变中酶催化位点的构象平衡”生物化学。

Koide,Shohei: "Conformational Equilibrium of an Enzymatic Catalytic Site in the Allosteric Transition." Biochemistry. 31. 5362-5368 (1992)

Koide,Shohei：“变构转变中酶催化位点的构象平衡”。