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.
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November 1993
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Research Article|
November 01 1993
Glucosamine-6-phosphate deaminase from Escherichia coli has a trimer of dimers structure with three intersubunit disulphides
M M Altamirano;
M M Altamirano
*Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, P.O. Box 70-159, Ciudad Universitaria, 04510, Mexico City, Mexico
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J A Plumbridge;
J A Plumbridge
†Institut de Biologie Physico-Chimique (CNRS, URA 1139), 75005, Paris, France
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H A Barba;
H A Barba
*Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, P.O. Box 70-159, Ciudad Universitaria, 04510, Mexico City, Mexico
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M L Calcagno
M L Calcagno
*Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, P.O. Box 70-159, Ciudad Universitaria, 04510, Mexico City, Mexico
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1993 The Biochemical Society, London
1993
Biochem J (1993) 295 (3): 645–648.
Citation
M M Altamirano, J A Plumbridge, H A Barba, M L Calcagno; Glucosamine-6-phosphate deaminase from Escherichia coli has a trimer of dimers structure with three intersubunit disulphides. Biochem J 1 November 1993; 295 (3): 645–648. doi: https://doi.org/10.1042/bj2950645
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