The pKa Value and Accessibility of Cysteine Residues Are Key Determinants for Protein Substrate Discrimination by Glutaredoxin

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• 作者：Kristine Steen Jensen ; Jeppe T. Pedersen ; Jakob R. Winther ; Kaare Teilum
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：April 22, 2014
• 年：2014
• 卷：53
• 期：15
• 页码：2533-2540
• 全文大小：404K
• 年卷期：v.53,no.15(April 22, 2014)
• ISSN：1520-4995

文摘

The enzyme glutaredoxin catalyzes glutathione exchange, but little is known about its interaction with protein substrates. Very different proteins are substrates in vitro, and the enzyme seems to have low requirements for specific protein interactions. Here we present a systematic investigation of the interaction between human glutaredoxin 1 and glutathionylated variants of a single model protein. Thus, single cysteine variants of acyl-coenzyme A binding protein were produced creating a set of substrates in the same protein background. The rate constants for deglutathionylation differ by more than 2 orders of magnitude between the best (k₁ = 1.75 脳 10⁵ M^鈥? s^鈥?) and the worst substrate (k₁ = 4 脳 10² M^鈥? s^鈥?). The pK_a values of the substrate cysteine residues were determined by NMR spectroscopy and found to vary from 8.2 to 9.9. Rates of glutaredoxin 1-catalyzed deglutathionylation were assessed with respect to substrate cysteine pK_a values, cysteine residue accessibility, local stability, and backbone dynamics. Good substrates are characterized by a combination of high accessibility of the glutathionylated site and low pK_a of the cysteine residue.