Denaturation and Reactivation of Dimeric Human Glutathione Reductase

Abstract

Human glutathione reductase (GR; which catalyzes the reaction NADPH + GSSG + H' -2 GSH + NADP') is an obligatory FAD-containing homodimer of known geometry. Native human GR, a potential target of antimalarial and cytostatic agents, cannot be dissociated by dilution or by means of subunitinterface mimetics, similarly to well-studied viral dimeric proteins. However, ab initio folding andlor dimerization of human GR can be inhibited by point mutations or by peptides corresponding to subunitinterface areas, for example synthetic peptide P I 1, which represents the intersubunit-contact helix HI 1. The structure of this peptide, which might assist inhibitor design, was solved by high-resolution NMR spectroscopy. Residues 440-453, were found to be a helical in the isolated peptide. To quantitate the efficacy of inhibitors such as P I 1, we developed the following unfolding/reactivation assay. The effects of various guanidine hydrochloride (Gdn/HCl) concentrations were studied by analytical ultracentrifugation. It was shown that human GR denatured by greater than 3 M Gdn/HCl is monomeric and free of FAD. Circular-dichroism experiments at 223 nm indicated a half-life of approximately 20 s at 20°C for the unfolding process. To optimize the reactivation yield, four parameters [protein concentration (x) in the range 0.3-10 pg/ml, cofactor supplementation, temperature (y; 0-32"C), and time (0-72 h)] were varied systematically, and a reactivation score z was given to each constellation of parameters. This type of analysis might be useful to optimize refolding and activation yields for other proteins. For human GR, the highest recovery was found not to occur at one of the corners of the x,y plain, but close to its center. Consequently, the optimal assay conditions for folding and dimerization inhibitors are as follows. The enzyme (at 300 pg/ml) is denatured by 5 M guanidine hydrochloride/5 mM dithiothreitol, then reactivated by dilution to 1 pg/ml at pH 6.9 and 20°C. In the absence of inhibitors, this procedure leads to 70% of the control activity within 8 h. Peptides representing the upper subunit interface (for instance residues 436-478) of human GR were found to inhibit refolding with EC,,,, values in the micromolar range, whereas fragments from other regions of the protein had no influence on this process. For peptide P11, the EC,,,, value was 20 pM. In conclusion, hGR, enzyme with a tight intersubunit contact area of 21 nm', appears to be suitable for studying protein folding, dimerization, and prosthetic-group complexation in the absence and presence of compounds that inhibit these processes. There is a shortage, at least for oligomeric enzymes of eukaryotes, of published systematic studies on protein (re)activation.Keywords: unfolding/reactivation ; human glutathione reductase; antiparasitic drug; protein-dimerization inhibitor; peptide-structure determination by NMR. Neuenheimer Feld 328, D-69120 Heidelberg, Germany. of glutathione, respectively ; Williams, 1992). Since each binding site for the substrate GSSG and each...

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