Protein Engineering Studies on Structure and Function of Amino Acid Dehydrogenase

氨基酸脱氢酶结构与功能的蛋白质工程研究

• 批准号: 02680159
• 负责人: TANIZAWA Katsuyuki
• 金额: $ 1.54万
• 依托单位: Grant-in-Aid for Scientific Research (c)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02680159/
• 关键词: Amino Acid Dehydrogenase; Chemical Modification; Pyridoxal Phosphate; Active Site; Site-directed Mutagenesis (6); ロイシン脱水素酵素; 耐熱性酵素; ピリドキサル5'ーリン酸; リジン残基

The following results have been obtained by the present studies.(a)Identification of an Active-Site Lysine in Leucine Dehydrogenase by Chemical Modification with Pyridoxal PhosphateLeucine dehydrogenase from Bacillus stearothermophilus was inactivated by incubation with pyridoxal 5'-phosphate(PLP)followed by reduction with sodium borohydride. The inactivation was completely retarded in the copresence of L-leucine and NAD^+. Several PLP molecules were indeed incorporated into one mol of the enzyme subunit concomitantly with the inactivation. Sequence analysis of the fluorescent peptides isolated from a proteolytic digest of the labeled protein revealed that Lys80, Lys91, Lys206, and Lys265 were labeled. Lys8O was most predominantly labeled and, in the presence of L-leucine and NAD^+, was specifically protected from the labeling. Furthermore, a linear relationship of about 1 : 1 was observed between the residual activity and the amount of PLP incorporated into Lys8O. Lys8O is conserved(b … More )Functional Analysis of Active-Site Lysine 80 in Leucine Dehydrogenase by Site-Directed MutagenesisLys80 of leucine dehydrogenase has been replaced by Ala, Arg, or Gln by sitedirected mutagenesis. The Lys8O mutant enzymes purified to homogeneity showed markedly low V values(0.2-1.6% of that of the wild-type enzyme)in the oxidative deamination, but variable_<max> values in the reductive amination(Lys8O-Ala, 89% ; Lys8O-Gln, 23% ; and Lys8O-Arg, 0.4% of that of the wild-type enzyme). The Lys8O-Ala and Lys8O-Gln mutant enzymes exhibited considerably increased K_m values for alpha-keto-iso-caproate, whereas the K_m values for NAD^+ and NADH of all the mutant enzymes were similar to those of the wild-type enzyme. iso-Caproate, a non-substrate analogue of alpha-keto-iso-caproate, competitively inhibited the reaction of both the wild-type and Lys8O-Ala mutant enzymes with K_i values much higher than the K_m value for alpha-keto-iso-caproate of the wild-type enzyme, suggesting the importance of an electrostatic interaction between the *-amino group of Lys8O and the alpha-carbonyl group of alpha-keto-iso-caproate in the binding of the keto acid substrate. Exogenously added primary amines including ammonium ion remarkably accelerated the reaction catalyzed by the Lys8OAla mutant enzyme, indicating that the added amines can partially replace the function of *-amino group of Lys8O depending on both their basicities (pK_a values) and molecular volumes. These results lead to the conclusion that the *-amino group of the active site Lys8O ftinctions as a general acid-base group in the catalysis of leucine dehyarogenase. Less

通过本研究获得了以下结果。（a）通过化学修饰与吡啶毒性磷酸甲甲甲基氨基糖酶从芽孢杆菌中鉴定在亮氨酸脱氢酶中的活性部位赖氨酸，通过与5'-磷酸盐（PLP）的吡啶多毒素（PL）在SADIUM上脱孵育，从而通过与SODODDRIDIM RORODIM DRODIM DORDIMDRIDEDIMDIMDRIDEDIUM抗生。在L-亮氨酸和nAD^+的共同点中，失活完全阻碍。确实将几种PLP分子与灭活同时掺入了一个mol的酶亚基中。从标记蛋白质的蛋白水解消化物中分离出的荧光肽的序列分析表明，标记了LYS80，LYS91，LYS206和LYS265。 Lys8O的标记最多，并且在L-达氨酸和NAD^+的存在下受到特异性保护。 Furthermore, a linear relationship of about 1 : 1 The Lys8O mutant enzymes purified to homogeneity shown markedly low V values(0.2-1.6% of that of the wild-type enzyme) in the oxidative deamination, but variable_<max> values ​​in the reduced amination(Lys8O-Ala, 89%; Lys8O-Gln​​, 23%; and Lys8O-Arg, 0.4%野生型酶的）。 lys8o-ala和lys8O-GLN突变酶仔细暴露的α-keto-iso-caproate的k_m值增加，而所有突变酶的NAD^+的k_m值与野生型酶的k_m值相似。等型甲状腺是α-酮-ISO-辅助酯的非基层类似物，竞争性抑制了野生型和lys8o-ala突变酶与K_I值的反应，而K_i值高于K_M高于k_m的k_m值，而k_m值则是Alpha-Keto-Iso-caprorate的Ensimantival antimentive antimentive antimentive antimentive and Intivition and and Intimentiation nigentive and Intimentiation and Anteriastion and Intimentive and *在酮酸底物的结合中，lys8O和α-酮 - 甲状腺丙酸α的α-羰基。外源添加的原发铵离子明显地加速了由lys8oala突变酶催化的反应，表明添加的铵基可以部分替代lys8O的 *-Amino lys8O组功能，这取决于其基本基础（PK_A值）和分子体积。这些结果得出的结论是，在亮氨酸脱氢酶催化中，活性位点lys8o ftinctions的 * - 氨基群是一般酸碱。较少的

项目成果

Katsuyuki Tanizawa: "Chemical and Kinetic Evidence for an Essential Lysine in the Thermostable Leucine Dehydrogenase from Bacillus stearothermophilus" Journal of Biological Chemistry. (1991)

Katsuyuki Tanizawa：“嗜热脂肪芽孢杆菌耐热亮氨酸脱氢酶中必需赖氨酸的化学和动力学证据”生物化学杂志。

Takahiro Matsuyama, Katsuyuki Tanizawa, Kenji Soda, and Toshio Fukui: "Leucine Dehydrogenase from Bacillus stearothermophilus : Identification of Active-Site Lysine Residue by Chemical Modification with Pyridoxal Phosphate." J. Biochem.(1992)

Takahiro Matsuyama、Katsuyuki Tanizawa、Kenji Soda 和 Toshio Fukui：“来自嗜热脂肪芽孢杆菌的亮氨酸脱氢酶：通过磷酸吡哆醛化学修饰鉴定活性位点赖氨酸残基。”

Takahiro Matsuyama: "Leucine Dehydrogenase from Bacillus stearothermophilus:Identification of ActiveーSite Lysine Residue by Chemical Modification with Pyridoxal Phosphate." J.Biochem.(1992)

Takahiro Matsuyama：“来自嗜热脂肪芽孢杆菌的亮氨酸脱氢酶：通过磷酸吡哆醛化学修饰鉴定活性位点赖氨酸残基。J.Biochem。”

Takahiro Matsuyama: "Leucine Dehydrogenase from Bacillus stearothermophilus:Identification of Active-Site Lysine Residue by Chemical Modification with Pyridoxal Phosphate." J.Biochem.(1992)

Takahiro Matsuyama：“来自嗜热脂肪芽孢杆菌的亮氨酸脱氢酶：通过磷酸吡哆醛化学修饰鉴定活性位点赖氨酸残基。”

Katsuyuki Tanizawa: "Chemical and Kinetic Evidevce for the Essential Lysine 80 in Levcine Dehydrogenase from Bacillus Stearothermophilu" J.Biol.Chem. (1992)

Katsuyuki Tanizawa：“嗜热脂肪芽孢杆菌 Levcine 脱氢酶中必需赖氨酸 80 的化学和动力学证据”J.Biol.Chem。