Synthesis and redox potential study of glutaredoxin 3 and selenocysteine-containing analogs

ORGN 640

Norman Metanis, metanis@scripps.edu1, Philip E. Dawson2, and Ehud Keinan1. (1) Department of Organic Chemistry, Technion - Israel Institute of Technology, Department of Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd. MB-20, La Jolla, CA 92037, (2) Department of Cell Biology and Chemistry, The Scripps Research Institute, 10550, N.Torrey Pines Rd. CVN 6, La Jolla, CA 92037
Sulfhydryl chemistry represents the focal point of all thiol-disulfide oxidoreductases of the thioredoxin superfamily, including thioredoxins (Trxs), and glutaredoxins (Grxs). These ubiquitous enzymes, which share a similar molecular architecture, known as the thioredoxin fold (α/β fold), perform a broad variety of cellular tasks. These enzymes also share the same active site motif (CXXC), where the pKa of the N-terminal Cys thiol is lower than that of free Cys. The tuning of the redox potential is partially determined by the geometry, and site-directed mutagenesis experiments have shown that radically changing the sequence can alter the redox potential of thioredoxin by up to 70 mV. Grx3 is a small (82 amino acids) thiol-disulfide oxidoreductase that acts as hydrogen donors to ribonucleotide reductase. This enzyme contains the active site sequence (CPYC) and its redox potential is -198 mV. Selenocysteine (Sec, U) can be viewed as the 21st amino acid in terms of ribosome-mediated protein synthesis. Most of the known selenoproteins are redox enzymes, in which the low pKa value of the selenol function (5.73 for Sec vs. 8.53 for Cys) confers unique biochemical properties. Determining the redox potential of the Sec-containing peptides and proteins is crucial for understanding its mechanism and substrate range. Four synthetic analogues of Grx3 have been synthesized: Grx3, Grx3(C11U), Grx3(C14U), and Grx3(C11U-C14U). All analogues were synthesized with Cys65Tyr, Met43Nle and Ala38carbamidomethylated-Cys (A38X) substitutions. The synthesis of Grx3 analogues was carried out by solid-phase peptide synthesis (SPPS) combined with Native Chemical Ligation (NCL) and selective alkylation of the Cys38 by iodoacetamide (Scheme bellow). The effect of these substitution on the redox potential of Grx3 will be discussed. The redox potentials of all Grx3 analogues were determined by direct protein-protein redox equilibria as described by Holmgren et al.1 1) Aslund, F.; Berndt, K.D.; Holmgren, A. J. Biol. Chem. 1997, 272, 30780-30786.

 

Proteins, Peptides, Amino Acids, and Enzyme Inhibitors
8:00 AM-12:00 PM, Thursday, 30 March 2006 Georgia World Congress Center -- C301, Oral

Division of Organic Chemistry

The 231st ACS National Meeting, Atlanta, GA, March 26-30, 2006