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Issue 1, 2009
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A residue outside the active site CXXC motif regulates the catalytic efficiency of Glutaredoxin 3

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Abstract

The glutaredoxin (Grx) family of oxidoreductases has a conserved residue at position 8 that varies between Arginine in Grx1 and Lysine in Grx3. It has been proposed that this Arg/Lys change is the main cause for the 35 mV difference in redox potential between the two enzymes. To gain insights into the catalytic machinery of Grx3 and directly evaluate the role of residue 8 in the catalysis of thioldisulfide exchange by this enzyme, we synthesized the “wild type” enzyme (sGrx3), and four analogues substituting the lysine at position 8 with arginine, ornithine (Orn), citrulline (Cit) and norvaline (Nva). The redox potential and equilibration kinetics with thioredoxin (Trx1) were determined for each enzyme by fluorescence intensity. While minor effects on redox potential were observed, we found that residue 8 had a more marked effect on the catalytic efficiency of this enzyme. Surprisingly, truncation of the functional group resulted in a more efficient enzyme, Lys8Nva, exhibiting rate constants that are an order of magnitude higher than sGrx3 for both forward and reverse reactions. These observations pose the question why would a residue that reduces the rate of enzyme turnover be evolutionarily conserved? The significant changes in the kinetic parameters suggest that this position plays an important role in the thioldisulfide exchange reaction by affecting the nucleophilicthiolate through electrostatic or hydrogen bonding interactions. Since the reduced Grx has an exposed thiol that could easily be alkylated, either Arg or Lys could act as a gatekeeper that deters unwanted electrophiles from attacking the active site thiolate.

Graphical abstract: A residue outside the active site CXXC motif regulates the catalytic efficiency of Glutaredoxin 3

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Publication details

The article was received on 01 Jul 2009, accepted on 24 Aug 2009 and first published on 22 Sep 2009


Article type: Paper
DOI: 10.1039/B912753D
Citation: Mol. BioSyst., 2009,6, 241-248
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    A residue outside the active site CXXC motif regulates the catalytic efficiency of Glutaredoxin 3

    T. Shekhter, N. Metanis, P. E. Dawson and E. Keinan, Mol. BioSyst., 2009, 6, 241
    DOI: 10.1039/B912753D

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