Construction and investigation of co-operativity in hybrids of norleucine- and glutamate-active subunits of clostridial glutamate dehydrogenase.

In vitro subunit hybridisation is a usehl technique for exploring inter-subunit interactions in oligomeric enzymes [I]. In order to explore the basis of allosteric bebaviour in glutamate dehydrogenase from Clmtridiurn syrnbimurn, hybrid hexamers of mutants, VIZ. triple mutant, K89L/A163G/S380A [2] and C320S [3] have been constructed. The mutant C320S is hUy active and shows normal allosteric properties but lacks the reactive cysteine. The triple mutant is active towards L-norleucine but not Lglutamate. Conditions have been established for unfolding and dissociating both these mutants and for refolding to form native hexamers. By renaturing mixtures of the two, hybrid hexamers containing 5 subunits of the triple mutant and 1 subunit of C320S were constructed and their co-operative behaviour has been investigated. The mutants K89UA163G/S380A and C320S (1 .5 mg/ml of each) were denatured separately at 2 5 T , by 4.5M and 4M urea respectively, for 60 and 30 min. The two denatured enzymes were mixed in 5 : 1 (triple mutant:C32OS) volume ratio, diluted 25-fold into 0.1 M potassium phosphate buffer (pH 7.0) containing 2 mM NAD+ and incubated at 25°C for 24 h. For both the mutants as much as 67% of the original enzyme activity was recovered after renaturation. The renatured mixture was concentrated and separated from NAD' by ion-exchange chromatography on a DEAE-Sephadex A-50 column and was eluted with 0.5 M NaCl in 0. I M potassium phosphate buffer (pH 7.0). The triple mutant, like the wild-type enzyme, is inactivated by Ellman's reagent, DTNB [I], which abolishes coenzyme binding. Thus, the DTNB-treated triple mutant should not bind to the NAD+-agarose column. The C320S mutant is not affected by DTNB, however, and does bind to the column. The refolded mixture of hybrid hexamers and triple mutant, both separately treated with DTNB, and the native triple mutant were each loaded on to the NAD+-agarose column and eluted with a gradient of NaCl. Only the native triple mutant binds to the column whereas