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

基本信息

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

项目摘要

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of Active-Site Lysine 80 in Leucine Dehydrogenase by Site-Directed MutagenesisLys80 of leucine dehydrogenase has been replaced by Ala, Arg, or Gln by sitedirected mutagenesis.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

目前的研究已获得以下结果。(a)通过磷酸吡哆醛化学修饰鉴定亮氨酸脱氢酶中的活性位点赖氨酸通过与5'-磷酸吡哆醛(PLP)温育然后还原来灭活来自嗜热脂肪芽孢杆菌的亮氨酸脱氢酶与硼氢化钠共存时，失活被完全延迟。 L-亮氨酸和 NAD^+ 确实在失活的同时掺入了 1 mol 酶亚基中。对从标记蛋白的蛋白水解消化物中分离出的荧光肽进行的序列分析显示，Lys80、Lys91、Lys206 和Lys265 被标记最多，L-亮氨酸和 NAD^+ 存在时，Lys8O 受到特别保护。此外，在残留活性与 Lys8O 中的 PLP 量之间观察到约 1:1 的线性关系。Lys8O 是保守的（b … 更多）通过位点定向对亮氨酸脱氢酶中的活性位点赖氨酸 80 进行功能分析。诱变亮氨酸脱氢酶的 Lys80 已通过定点诱变被 Ala、Arg 或 Gln 取代 Lys8O 突变体。纯化至均质的酶在氧化脱氨中表现出明显较低的 V 值（野生型酶的 0.2-1.6%），但在还原氨化中表现出可变_<max>值（Lys8O-Ala，89%；Lys8O- Gln，23%；Lys8O-Arg，野生型酶的 0.4%）。 Lys8O-Gln 突变酶对 α-酮基异己酸的 K_m 值显着增加，而所有突变酶的 NAD^+ 和 NADH 的 K_m 值与野生型酶相似iso-Caproate 是 alpha-keto-iso-caproate 的非底物类似物，竞争性抑制野生型和 Lys8O-Ala 突变酶的反应，K_i 值远高于。野生型酶的α-酮异己酸的K_m值，表明Lys8O的*-氨基和α-酮异己酸的α-羰基之间静电相互作用在结合中的重要性外源添加的伯胺（包括加速铵离子）显着地促进了Lys8OAla突变酶的催化反应，表明添加的胺可以部分替代酮酸底物的功能。 Lys8O 的*-氨基取决于其碱度（pK_a 值）和分子体积。这些结果得出这样的结论：Lys8O 活性位点的*-氨基在亮氨酸脱氢酶的催化中充当通用酸碱基团。。较少的