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 mutagenesis.
The nucleotide sequence of the nagB gene in Escherichia coli, encoding glucosamine-6-phosphate deaminase, located four cysteinyl residues at positions 118, 219, 228, and 239. Chemical modification studies performed with the purified enzyme had shown that the sulfhydryl groups of two of these residues form a vicinal pair in the enzyme and are easily modified by thiol reagents. The allosteric transition to the more active conformer (R), produced by the binding of homotropic (D-glucosamine 6-phosphate or 2-deoxy-2-amino-D-glucitol 6-phosphate) or heterotropic (N-acetyl-D-glucosamine 6-phosphate) ligands, completely protected these thiols against chemical modification. Selective cyanylation of the vicinal thiols with 2-nitro-5-(thiocyanato)benzoate, followed by alkaline hydrolysis to produce chain cleavage at the modified cysteines, gave a pattern of polypeptides which allowed us to identify Cys118 and Cys239 as the residues forming the thiol pair. Subsequently, three mutated forms of the gene were constructed by oligonucleotide-directed mutagenesis, in which one or both of the cysteine codons were changed to serine. The mutant proteins were overexpressed and purified, and their kinetics were studied. The dithiol formed by Cys118 and Cys239 was necessary for maximum catalytic activity. The single replacements and the double mutation affected catalytic efficiency in a similar way, which was also identical to the effect of the chemical block of the thiol pair. However, only one of these cysteinyl residues, Cys239, had a significant role in the allosteric transition, and its substitution for serine reduced the allosteric interaction energy, due to a lower value of KT.
Altamirano
M M
MM
Departamento de Bioquimica, Facultad de Medicina, Universidad Nacional Autónoma de México, D.F.
Plumbridge
J A
JA
Calcagno
M L
ML
D019747:Aldose-Ketose Isomerases; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D015032:Zinc / Q000494:pharmacology
D013438:Sulfhydryl Compounds; C020582:glucosamine-6-phosphate isomerase; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; D015032:Zinc; D003545:Cysteine