Glucosamine-6-phosphate deaminase from Escherichia coli has a trimer of dimers structure with three intersubunit disulphides
- 1 November 1993
- journal article
- Published by Portland Press Ltd. in Biochemical Journal
- Vol. 295 (3), 645-648
- https://doi.org/10.1042/bj2950645
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.Keywords
This publication has 11 references indexed in Scilit:
- Crystallization and preliminary crystallographic studies of glucosamine-6-phosphate deaminase from Escherichia coli K12Journal of Molecular Biology, 1992
- Identification of two cysteine residues forming a pair of vicinal thiols in glucosamine-6-phosphate deaminase from Escherichia coli and a study of their functional role by site-directed mutagenesisBiochemistry, 1992
- Repression and induction of the nag regulon of Escherichia coll K‐12: the roles of nagC and nagA in maintenance of the uninduced stateMolecular Microbiology, 1991
- Secondary structure of Escherichia coli glucosamine-6-phosphate deaminase from amino acid sequence and circular dichroism spectroscopyBiochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1991
- Nucleotide sequences of the Escherichia coli nagE and nagB genes: the structural genes for the N-acetylglucosamine transport protein of the bacterial phosphoenolpyruvate: sugar phosphotrans-ferase system and for glucosamine-6-phosphate deaminaseGene, 1988
- Sulfhydryl groups of glucosamine-6-phosphate isomerase deaminase from Escherichia coliArchives of Biochemistry and Biophysics, 1987
- Quantitation of proteins by elution of Coomassie brilliant blue R from stained bands after sodium dodecyl sulfate-polyacrylamide gel electrophoresisAnalytical Biochemistry, 1986
- Purification, molecular and kinetic properties of glucosamine-6-phosphate isomerase (deaminase) from Escherichia coliBiochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1984
- Electrophoretic analysis of the unfolding of proteins by ureaJournal of Molecular Biology, 1979
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970