title: Glucosamine-6-phosphate deaminase from Escherichia coli has a trimer of dimers structure with three intersubunit disulphides.
abstract: Glucosamine-6-phosphate deaminase is an oligomeric protein composed of six identical 29.7 kDa subunits. Each subunit has four cysteine residues located at positions 118, 219, 228 and 239. We have previously shown that Cys-118 and Cys-239 form a pair of vicinal thiols, the reactivity of which changes with the allosteric transition. The site-directed mutations Cys-->Ser corresponding to the other two cysteine residues have been constructed, as well as some selected multiple mutations involving the four cysteines. Thiol and disulphide measurements on the wild-type and mutant enzymes indicate that thiols from Cys-219 are oxidized and form interchain disulphide bonds. The disulphide-linked dimer was demonstrated by SDS/PAGE. This result is consistent with preliminary crystallographic data and thermal denaturation studies, and strongly suggests that glucosamine-6-phosphate deaminase is a trimer of disulphide-linked dimers. The mutant forms of the deaminase lacking the interchain disulphide bond or the thiol at Cys-228 are both stable hexamers showing the same sensitivity to urea denaturation as the wild-type protein. Furthermore, these Cys-->Ser mutants display the same kinetics and allosteric properties as those already described for the wild-type enzyme.
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad Universitaria, Mexico City.
authors.forename: M M
authors.initials: MM
authors.lastname: Altamirano
authors.forename: J A
authors.initials: JA
authors.lastname: Plumbridge
authors.forename: H A
authors.initials: HA
authors.lastname: Barba
authors.forename: M L
authors.initials: ML
authors.lastname: Calcagno
mesh_terms: D019747:Aldose-Ketose Isomerases; D000494:Allosteric Regulation; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation
chemical_list: D004220:Disulfides; D046911:Macromolecular Substances; C020582:glucosamine-6-phosphate isomerase; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; D003545:Cysteine