Studies on Active Site of Transaminase by Site-Directed Mutagenesis

转氨酶活性位点定点诱变研究

• 批准号: 01480524
• 负责人: KAGAMIYAMA Hiroyuki
• 金额: $ 4.16万
• 依托单位: Osaka Medical College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-01480524/
• 关键词: Mutagenesis; Transaminase; Aspartate Aminotransferase; Enzyme Mechanism; Pyridoxal Phosphate; Active Site; Protein Engineering; 部位特異性突然変異; アスパラギン酸アミノ基転移酵素; ピリドキサル燐酸; 活性中心; 酵素の活性中心; 基質認識

We have examined the functional role of some active site residues of E. coli aspartate aminotransferase by using kinetic analysis on mutant enzymes produced by the site-directed mutagenesis. Aspartate aminotransferase has been the most extensively studied representative of many transaminases ; X-ray analysis has been done. Our findings are as follows :1. Lys258 is essential for catalysis, acting as a catalytic base to withdraw an a -proton from the amino acid substrate, which is a prerequisite for the catalysis.2. A negative charge at position 222, and a hydrogen bond between the hydroxyl group of Tyr225 and the unprotonated hydroxyl group of the coenzyme probably help in lowering the electron density of the coenzyme to facilitate the a- proton removal.3. Arg292 and Arg386 are essential for the recognition of the dicarboxylic substrates.4. Substitution of Arg292 to uncharged residues greatly enhanced the catalytic efficiency of the transamination of neutral amino acid without. any effect on the binding.5. The endole ring of Trpl4O not only regulates the rotational movement of the coenzyme ring during the catalysis, but it may be also involved in the binding of the carboxyl side chain of the dicarboxylic acid substrates.6. The phenol group of Tyr7O is essential for the stabilization of the transition states with all substrates. The presence of the benzen ring at position 70 is necessary to recognize the glutanate-2-oxoglutarate substrate pair.

我们研究了E.大肠杆菌天冬氨酸氨基转移酶的酶动力学分析。天冬氨酸氨基转移酶是许多转氨酶中研究最广泛的代表，已进行了X射线分析。研究结果如下：1. Lys 258是催化所必需的，作为催化碱从氨基酸底物中提取a -质子，这是催化的先决条件. 222位的负电荷以及Tyr 225的羟基与辅酶的未质子化羟基之间的氢键可能有助于降低辅酶的电子密度以促进α-质子的去除。Arg 292和Arg 386是识别二羧酸底物所必需的. Arg 292被不带电荷的残基取代后，大大提高了中性氨基酸转氨反应的催化效率。对约束力的任何影响。Trp 14 O的内孔环不仅在催化过程中调节辅酶环的旋转运动，而且还可能参与二羧酸底物羧基侧链的结合. Tyr 7 O的酚基对于所有底物的过渡态的稳定是必不可少的。在位置70处存在苯环对于识别戊二酸-2-酮戊二酸底物对是必要的。

项目成果

Hayashi,H.: "〔Arg292--Val〕or 〔Arg292--Leu〕 Mutation Enhances The Reactivity of Escherichia coli Aspartate Aminotransferase with Aromatic Amino Acids." Biochem.Biophys.REs.Commun.159. 337-342 (1989)

Hayashi, H.：“[Arg292--Val] 或 [Arg292--Leu] 突变增强了大肠杆菌天冬氨酸转氨酶与芳香氨基酸的反应性。Biochem.Biophys.REs.Commun.159 (1989)。

Hayashi,H.: "Effect of Replacement of Tryptophan-140 by Phenylalanine or Glycine on The Function of Escherichia coli Aspartate Aminotransferase." Biochem.,Biophys.Rse.Commun.167. 407-412 (1990)

Hayashi,H.：“用苯丙氨酸或甘氨酸替代色氨酸-140 对大肠杆菌天冬氨酸转氨酶功能的影响。”

Morino,Y.: "Mammalian Aspartate Aminotransferase Isozymes:From DNA to Protein." Annals of The New York Academy of Sciences. 585. 32-47 (1990)

Morino,Y.：“哺乳动物天冬氨酸转氨酶同工酶：从 DNA 到蛋白质。”

Morino, Y.: "Mammalian Aspartate Aminotransferase Isozymes : From DNA to Protein." Annals of The Nwe York Academy of Sciences. 585. 32-47 (1990)

Morino, Y.：“哺乳动物天冬氨酸转氨酶同工酶：从 DNA 到蛋白质。”

Takato Yano: "The Role of His^<143> in the Catalytic Mechanism of Escherichia coli Aspartate Aminotransferase" J.Biol.Chem.266. 6079-6085 (1991)

Takato Yano：“His^<143> 在大肠杆菌天冬氨酸转氨酶催化机制中的作用”J.Biol.Chem.266。