Site-specific mutagenesis of lysine-204, tyrosine-224, tyrosine-228, and histidine-307 of porcine kidney D-amino acid oxidase and the implications as to its catalytic function.
Site-specific mutagenesis of lysine-204, tyrosine-224, tyrosine-228, and histidine-307 of porcine kidney D-amino acid oxidase and the implications as to its catalytic function.
复制标题
猪肾 D-氨基酸氧化酶赖氨酸 204、酪氨酸 224、酪氨酸 228 和组氨酸 307 的定点突变及其催化功能的影响。
DOI：
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发表时间：
1989
期刊：
影响因子：
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通讯作者：
Y. Miyake
中科院分区：
文献类型：
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作者：
F. Watanabe;K. Fukui;K. Momoi;Y. Miyake
为了评估猪肾D-氨基酸氧化酶[EC 1.4.3.3] (DAO)中Lys-204、Tyr-224、Tyr-228和His-307对其催化功能的可能贡献，我们通过寡核苷酸指导在体外构建了编码具有Glu-204、Phe-224、Phe-228和Leu-307的酶的四个点突变cDNA。诱变。四种突变型 cDNA 和野生型 cDNA 可以以类似的效率在体外表达，并且从 5 微克各自的加帽 RNA 中产生约 200 ng 的每种酶蛋白。体外合成的突变酶在SDS-聚丙烯酰胺凝胶上的电泳迁移率与野生型DAO几乎相同，计算分子量为38,000。 Glu-204 和 Phe-224 突变体 DAO 显示出与野生型酶相当的酶活性，并且受到 DAO 的有效竞争性抑制剂苯甲酸钠的强烈抑制。两种突变型 DAO 的动力学参数也与野生型 DAO 相当。另一方面，Phe-228 和 Leu-307 突变体 DAO 没有表现出可检测到的活性。结果表明，Tyr-228和His-307对活性位点的构成起重要作用或直接参与反应，而Lys-204和Tyr-224在酶反应中不是必需的。
In order to evaluate the possible contributions of Lys-204, Tyr-224, Tyr-228, and His-307 in porcine kidney D-amino acid oxidase [EC 1.4.3.3] (DAO) to its catalytic function, we constructed four point mutant cDNAs encoding enzymes possessing Glu-204, Phe-224, Phe-228, and Leu-307 by oligonucleotide-directed in vitro mutagenesis. The four mutant cDNAs and the wild type cDNA could be expressed in vitro with similar efficiencies and about 200 ng of each enzyme protein was produced from 5 micrograms of the respective capped RNA. The electrophoretic mobilities of the in vitro synthesized mutant enzymes on SDS-polyacrylamide gel were almost identical with that of the wild type DAO, and the molecular weight was calculated to be 38,000. The Glu-204 and Phe-224 mutant DAOs showed comparable enzyme activities to that of the wild type enzyme, and were inhibited strongly by sodium benzoate, a potent competitive inhibitor of DAO. The kinetic parameters of the two mutant DAOs were also comparable to those of the wild type DAO. On the other hand, the Phe-228 and Leu-307 mutant DAOs showed no detectable activity. The results indicate that Tyr-228 and His-307 play important roles as to the constitution of the active site or participate in the reaction directly, while Lys-204 and Tyr-224 are not essential in the enzyme reaction.