Construction and characterization of mutant water-soluble PQQ glucose dehydrogenases with altered Km values -: Site-directed mutagenesis studies on the putative active site
Construction and characterization of mutant water-soluble PQQ glucose dehydrogenases with altered Km values -: Site-directed mutagenesis studies on the putative active site
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DOI：
10.1006/bbrc.1999.1157
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发表时间：
1999-11-02
影响因子：
3.1
通讯作者：
Sode, K
中科院分区：
文献类型：
--
作者：
Igarashi, S;Ohtera, T;Sode, K
以含有吡咯喹啉基团的水溶性葡萄糖脱氢酶突变酶PQQGDH-B为辅基，进行了定点突变研究。与野生型酶相比，Glu277残基被Gly取代后，葡萄糖的K-m值降低，底物专一性改变。对Glu277的邻近氨基酸残基进行了突变分析，构建了Asp275Glu、Asp276Glu、Ile278Phe和Asn279His。考虑到Asp275Glu、Asp276Glu和Glu277Gly表现出对EDTA耐受性的急剧下降，该区域可能构建了一个PQQGDH-B推测的活性部位，例如负责结合PQQ的钙结合部位。构建了一系列由Ala、Val、Asp、Asn、His、Gin或To Lys取代的Glu277变异体，它们都表现出K-m值降低和底物专一性改变。其中，Glu277Lys具有与野生型相似的热稳定性，但其催化效率明显高于野生型。本文还讨论了Glu277Lys在分析中的潜在应用。(C)1999年学术出版社。
Based on a PCR mutant enzyme of water-soluble glucose dehydrogenase-harboring pyrroloquinoline quinone as the prosthetic group, PQQGDH-B, a site-directed mutagenesis study was carried out. The substitution of Glu277 residue with Gly resulted in a decrease in the K-m value for glucose and altered the substrate specificity profile, compared with the wildtype enzyme. Mutational analyses on the neighboring amino acid residues of Glu277 were also carried out and constructed Asp275Glu, Asp276Glu, Ile278Phe, and Asn279His. Considering that Asp275Glu, Asp276Glu and also Glu277Gly showed drastic decreases in EDTA tolerance, this region may construct a PQQGDH-B putative active site, such as a binding site for Ca2+, which is responsible for the binding PQQ A series of Glu277 variants, Glu277 substituted by Ala, Val, Asp, Asn, His, Gin, or to Lys, was constructed and they all showed decreased K-m values and altered substrate specificity profiles. Among them, Glu277Lys showed similar thermal stability with the wild-type enzyme, but its catalytic efficiency increased significantly, compared with the wild-type enzyme. The potential applications of Glu277Lys in analytical use are also discussed. (C) 1999 Academic Press.