Kuwahara Mitsuhiko, Tamura Takashi, Kawamura Kentaro, Inagaki Kenji
Department of Bioresources Chemistry, Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan.
Biosci Biotechnol Biochem. 2011;75(3):516-21. doi: 10.1271/bbb.100740. Epub 2011 Mar 7.
Mammalian thioredoxin reductases (TrxRs) contain selenium as selenocysteine (Sec) in the C-terminal redox center -Gly-Cys-Sec-Gly-OH to reduce Trx and other substrates; a Sec-to-Cys substitution in mammalian TrxR yields an almost inactive enzyme. The corresponding tetrapeptide sequence in Drosophila melanogaster TrxR (Dm-TrxR), -Ser-Cys-Cys-Ser-OH, endows the orthologous enzyme with a catalytic competence similar to mammalian selenoenzymes, but implementation of the Ser-containing tetrapeptide sequence SCCS into the mammalian enzyme does not restore the activity of the Sec-to-Cys mutant form (turnover number <2/min). MOPAC calculation suggested that the C-terminal hexapeptide Pro-Ala-Ser-Cys-Cys-Ser-OH functions as a redox center that alleviates the necessity for selenium in Dm-TrxR, and a mutant form of human lung TrxR that mimics this hexapeptide sequence showed improved catalytic turnover (17.4/min for DTNB and 13.2/min for E. coli trx) compared to the Sec-to-Cys mutant. MOPAC calculation also suggested that the dominant form of the Pro-containing hexapeptide is a C+ conformation, which perhaps has a catalytic advantage in facile reduction of the intramolecular disulfide bond between Cys497 and Cys498 by the N-terminal redox center in the neighboring subunit.
哺乳动物硫氧还蛋白还原酶（TrxRs）在其C端氧化还原中心-Gly-Cys-Sec-Gly-OH中含有作为硒代半胱氨酸（Sec）的硒，以还原Trx和其他底物；哺乳动物TrxR中Sec被Cys取代会产生一种几乎无活性的酶。果蝇TrxR（Dm-TrxR）中的相应四肽序列-Ser-Cys-Cys-Ser-OH，赋予直系同源酶与哺乳动物硒酶相似的催化能力，但将含Ser的四肽序列SCCS引入哺乳动物酶中并不能恢复Sec-to-Cys突变体形式的活性（周转数<2/分钟）。MOPAC计算表明，C端六肽Pro-Ala-Ser-Cys-Cys-Ser-OH作为氧化还原中心，减轻了Dm-TrxR中对硒的需求，与Sec-to-Cys突变体相比，模仿该六肽序列的人肺TrxR突变体形式显示出改善的催化周转（对DTNB为17.4/分钟，对大肠杆菌trx为13.2/分钟）。MOPAC计算还表明，含Pro的六肽的主要形式是C+构象，这可能在相邻亚基的N端氧化还原中心轻松还原Cys497和Cys498之间的分子内二硫键方面具有催化优势。
