Glutamate dehydrogenase from Clostridium symbiosum has two cysteine residues, C144 and C320. The single mutant C320S and a double mutant with both cysteines replaced by serine have been compared with one another in terms of long-term stability and other properties. Specific activities and kinetic parameters were relatively little affected, but stability was improved-e.g. at 25 degrees C sterile, sealed samples of wild-type enzyme, C320S and the double mutant at 0.1 mg/ml in 0.1 M phosphate buffer, pH 7 lost 50%, 42% and 32% of activity over 60 days. For the first two proteins this loss was partly reversible with dithiothreitol. When wild-type enzyme was deliberately contaminated with 1 mu M Cu2+ it became less stable and formed aggregates, whereas the double mutant was not affected. The double mutation thus removes a source of instability through -SH oxidation that would be accentuated by any heavy metal contamination of solutions. (C) 2007 Elsevier Inc. All rights reserved.