GLUCOSAMINE-6-PHOSPHATE DEAMINASE FROM ESCHERICHIA-COLI HAS A TRIMER OF DIMERS STRUCTURE WITH 3 INTERSUBUNIT DISULFIDES

Glucosamine-6-phosphate deaminase is an oligomeric protein composed of six identical 29.7 kDa subunits. Each subunit has four cysteine resid ues located at positions 118, 219, 228 and 239. We have previously sho wn that Cys-1 18 and Cys-239 form a pair of vicinal thiols, the reacti vity of which changes with the allosteric transition. The site-directe d mutations Cys-->Ser corresponding to the other two cysteine residues have been constructed, as well as some selected multiple mutations in volving the four cysteines. Thiol and disulphide measurements on the w ild-type and mutant enzymes indicate that thiols from Cys-219 are oxid ized and form interchain disulphide bonds. The disulphide-linked dimer was demonstrated by SDS/PAGE. This result is consistent with prelimin ary crystallographic data and thermal denaturation studies, and strong ly suggests that glucosamine-6-phosphate deaminase is a trimer of disu lphide-linked dimers. The mutant forms of the deaminase lacking the in terchain disulphide bond or the thiol at Cys-228 are both stable hexam ers showing the same sensitivity to urea denaturation as the wild-type protein. Furthermore, these Cys-->Ser mutants display the same kineti cs and allosteric properties as those already described for the wild-t ype enzyme.