Investigation of the C-Terminal Redox Center of High-Mr Thioredoxin Reductase by Protein Engineering and Semisynthesis

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• 作者：Brian E. Eckenroth ; Brian M. Lacey ; Adam P. Lothrop ; Katharine M. Harris ; Robert J. Hondal
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：August 21, 2007
• 年：2007
• 卷：46
• 期：33
• 页码：9472 - 9483
• 全文大小：205K
• 年卷期：v.46,no.33(August 21, 2007)
• ISSN：1520-4995

文摘

High-molecular weight thioredoxin reductases (TRs) catalyze the reduction of the redox-activedisulfide bond of thioredoxin, but an important difference in the TR family is the sequence of the C-terminalredox-active tetrapeptide that interacts directly with thioredoxin, especially the presence or absence of aselenocysteine (Sec) residue in this tetrapeptide. In this study, we have employed protein engineeringtechniques to investigate the C-terminal redox-active tetrapeptides of three different TRs: mousemitochondrial TR (mTR3), Drosophila melanogaster TR (DmTR), and the mitochondrial TR fromCaenorhabditis elegans (CeTR2), which have C-terminal tetrapeptide sequences of Gly-Cys-Sec-Gly,Ser-Cys-Cys-Ser, and Gly-Cys-Cys-Gly, respectively. Three different types of mutations and chemicalmodifications were performed in this study: insertion of alanine residues between the cysteine residuesof the Cys-Cys or Cys-Sec dyads, modification of the charge at the C-terminus, and altering the positionof the Sec residue in the mammalian enzyme. The results show that mTR3 is quite accommodating toinsertion of alanine residues into the Cys-Sec dyad, with only a 4-6-fold drop in catalytic activity. Incontrast, the activity of both DmTR and CeTR2 was reduced 100-300-fold when alanine residues wereinserted into the Cys-Cys dyad. We have tested the importance of a salt bridge between the C-terminusand a basic residue that was proposed for orienting the Cys-Sec dyad of mTR3 for proper catalytic positionby changing the C-terminal carboxylate to a carboxamide. The result is an enzyme with twice the activityas the wild-type mammalian enzyme. A similar result was achieved when the C-terminal carboxylate ofDmTR was converted to a hydroxamic acid or a thiocarboxylate. Last, reversing the positions of the Cysand Sec residues in the catalytic dyad resulted in a 100-fold loss of catalytic activity. Taken together, theresults support our previous model of Sec as the leaving group during reduction of the C-terminus duringthe catalytic cycle.