Studies on the Novel Enzymatie Reaction Mechanisms Taking into Account the Enzyme Fluctuations

考虑酶波动的新型酶反应机制研究

• 批准号: 12680634
• 负责人: KAWAI Yasushi
• 金额: $ 2.37万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680634/
• 关键词: hydrolase; reaction mechanism; viscosity effect; α-chymotrypsin; fluctuation; reductase

In order to clarify the significance of conformational fluctuation of an enzyme in the catalytic mechanism, the dependence of rate constants for hydrolyses catalyzed by various serine proteases on medium viscosity were investigated. Acylation of α-chymotrypsin by a p-nitroanilides is hardly affected by medium viscosity, while that by a p-nitrophenyl ester decreases with increase in medium viscosity. This tendency is also observed in β-trypsin-catalyzed hydrolyses. For these enzymes, the conformational effect of the enzyme exists in formation of a tetrahedral intermedite, whereas its breakdown is almost free from the conformational effect. On the other hand, in addition to the acylation of subtilisin Carlsberg by p-nitrophenyl esters, that by p-nitroanilides is also influenced by medium viscosity. This suggests that both formation of (ES)_T and following breakdown of it are assisted by the conformational change of the enzyme. These results reveal that, although serine proteases have the same catalytic mechanism each other, there have some differences in the conformational effects during the catalytic processes.In order to extend this method to the enzymes other than proteases, several novel reductases were isolated and purified from the cells of baker's yeast, which catalyze the asymmetric reduction of a carbon-carbon double bond in nitroalkenes and α,β-unsaturated ketones.

为了阐明酶的构象波动在催化机理中的重要性，研究了不同丝氨酸蛋白酶催化水解反应的速率常数对介质粘度的依赖性。对硝基苯胺酰化α-糜蛋白酶的反应受介质粘度的影响不大，而对硝基苯酯酰化α-糜蛋白酶的反应则随介质粘度的增加而降低。在β-胰蛋白酶催化的水解中也观察到这种趋势。对于这些酶，酶的构象效应存在于四面体中间体的形成中，而其分解几乎不受构象效应的影响。另一方面，除了对硝基苯基酯对枯草杆菌蛋白酶的酰化作用外，介质粘度对对硝基苯胺酰化枯草杆菌蛋白酶的酰化作用也有影响。这表明（ES）_T的形成和随后的分解都是由酶的构象变化所辅助的。这些结果表明，虽然丝氨酸蛋白酶具有相同的催化机理，但在催化过程中的构象效应存在一定的差异，为了将这种方法推广到蛋白酶以外的酶，我们从面包酵母细胞中分离纯化了几种新的还原酶，它们催化硝基烯烃和α，β-不饱和酮的碳-碳双键的不对称还原。

项目成果

Yasushi Kawai: "Asymmetric synthesis of α-chiral ketones by the reduction of enones with baker's yeast"Tetrahedron : Asymmetry. 12. 3007-3013 (2001)

Yasushi Kawai：“通过用面包酵母还原烯酮来不对称合成 α-手性酮” 四面体：不对称。 12. 3007-3013 (2001)

Yasushi Kawai: "6-Chloro-2, 4-dinitrophenylhdrazine as a useful crystalline agent for the determination of absolute configuration"Tetrahedron Letters. 43・3. 465-467 (2002)

Yasushi Kawai：“6-Chloro-2, 4-二硝基苯肼作为确定绝对构型的有用结晶剂”Tetrahedron Letters 43・3 (2002)。

Yasushi Kawai: "Asymmetric synthesis of a nitroalkane by the use of novel nitroalkane reductase from baker's yeast"Tetrahedron Letters. 42. 3367-3368 (2001)

Yasushi Kawai：“使用来自面包酵母的新型硝基烷还原酶不对称合成硝基烷”四面体快报。

Yasushi Kawai: "6-Chloro-2,4-dinitrophenylhydrazine as a useful crystalline agent for the determination of absolute configuration"Tetrahedron Letters. 43. 465-467 (2002)

Yasushi Kawai：“6-氯-2,4-二硝基苯肼作为一种有用的结晶剂，用于测定绝对构型”四面体快报。

Yasushi Kawai: "Asymmetric Synthesis of a nitroalkane by the use of novel nitroalkane reductase from baker's yeast"Tetrahedron Letters. (in press). (2001)

Yasushi Kawai：“使用来自面包酵母的新型硝基烷还原酶不对称合成硝基烷”四面体快报。