Construction of a dimeric form of glutamate dehydrogenase from Clostridium symbiosum by site-directed mutagenesis.

By using site-directed mutagenesis, Phe-187, one of the amino-acid residues involved in hydrophobic interaction between the three identical dimers comprising the hexamer of Clostridium symbiosum glutamate dehydrogenase (GDH), has been replaced by an aspartic acid residue. Over-expression in Escherichia coli led to production of large amounts of a soluble protein which, though devoid of GDH activity, showed the expected subunit M(r) on SDS-PAGE, and cross-reacted with an anti-GDH antibody preparation in Western blots. The antibody was used to monitor purification of the inactive protein. F187D GDH showed altered mobility on non-denaturing electrophoresis, consistent with changed size and/or surface charge. Gel filtration on a calibrated column indicated an M(r) of 87000 +/- 3000. The mutant enzyme did not bind to the dye column routinely used in preparing wild-type GDH. Nevertheless suspicions of major misfolding were allayed by the results of chemical modification studies: as with wild-type GDH, NAD+ completely protected one-SH group against modification by DTNB, implying normal coenzyme binding. A significant difference, however, is that in the mutant enzyme both cysteine groups were modified by DTNB, rather than C320 only. The CD spectrum in the far-UV region indicated no major change in secondary structure in the mutant protein. The near-UV CD spectrum, however, was less intense and showed a pronounced Phe contribution, possibly reflecting the changed environment of Phe-199, which would be buried in the hexamer. Sedimentation velocity experiments gave corrected coefficients S20,W of 11.08 S and 5.29 S for the wild-type and mutant proteins. Sedimentation equilibrium gave weight average molar masses M(r,app) of 280000 +/- 5000 g/mol. consistent with the hexameric structure for the wild-type protein and 135000 +/- 3000 g/mol for F187D. The value for the mutant is intermediate between the values expected for a dimer (98000) and a trimer (147000). To investigate the basis of this, sedimentation equilibrium experiments were performed over a range of protein concentrations. M(r,app) showed a linear dependence on concentration and a value of 108 118 g/mol at infinite dilution. This indicates a rapid equilibrium between dimeric and hexameric forms of the mutant protein with an equilibrium constant of 0.13 l/g. An independent analysis of the radial absorption scans with Microcal Origin software indicated a threefold association constant of 0.11 l/g. Introduction of the F187D mutation thus appears to have been successful in producing a dimeric GDH species. Since this protein is inactive it is possible that activity requires subunit interaction around the 3-fold symmetry axis. On the other hand this mutation may disrupt the structure in a way that cannot be extrapolated to other dimers. This issue can only be resolved by making alternative dimeric mutants.