A MONOMERIC MUTANT OF CLOSTRIDIUM-SYMBIOSUM GLUTAMATE-DEHYDROGENASE -COMPARISON WITH A STRUCTURED MONOMERIC INTERMEDIATE OBTAINED DURING REFOLDING

The refolding of Clostridium symbiosum glutamate dehydrogenase (GDH) i nvolves the formation of an inactive structured monomeric intermediate prior to its concentration-dependent association. The structured mono mer obtained after removal of guanidinium chloride was stable and comp etent for reconstitution into active hexamers. Site-directed mutagenes is of C. symbiosum gdh gene was performed to replace the residues Arg- 61 and Phe-187 which are involved in subunit-subunit interactions, as determined by three-dimensional structure analysis. Heterologous over- expression in Escherichia coli of the double mutant (R61E/F187D) led t o the production of a soluble protein with a molecular mass consistent with the monomeric form of clostridial GDH. This protein is catalytic ally inactive but cross-reacts with an anti-wild-type GDH antibody pre paration. The double mutant R61E/F187D does not assemble into hexamers . The physical properties and the stability toward guanidinium chlorid e and urea of R61E/F187D were studied and compared to those of the str uctured monomeric intermediate.