Evidence for vicinal thiols and their functional role in glucosamine-6-phosphate deaminase from Escherichia coli.
Methylation of glucosamine-6-phosphate isomerase deaminase (2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase, deaminating, or glucosamine-6-phosphate deaminase, EC 5.3.1.10), from Escherichia coli produces a modified protein having two alkylated sulfhydryls per each polypeptide chain. The enzyme is still active and allosteric, but exhibits a lower homotropic cooperativity and its Vmax/Etotal is almost exactly half that of the native enzyme. Arsenite produces comparable kinetic changes that can be reversed with ethanedithiol but not with 2-thioethanol or dialysis. Thiols can be oxidized by molecular oxygen using the (1,10-phenanthroline)3-Cu(II) complex as catalyst; the enzyme obtained no longer has titrable SH groups with 5,5'-dithiobis(2-nitrobenzoic acid) and displays kinetic behavior similar to that of the other chemically modified forms of the deaminase using monofunctional or bifunctional reagents. The results reported indicate that the involved sulfhydryls are vicinal groups, and are located in a region of the molecule that moves as a whole in the allosteric transition.
Altamirano
M M
MM
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico.
Libreros-Minotta
C A
CA
Lara-Lemus
R
R
Calcagno
M
M
D019747:Aldose-Ketose Isomerases; D001151:Arsenic / Q000494:pharmacology; D018053:Arsenites; D002238:Carbohydrate Epimerases / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008745:Methylation; D010618:Phenanthrolines / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000378:metabolism
D018053:Arsenites; D003432:Cross-Linking Reagents; D010618:Phenanthrolines; D013438:Sulfhydryl Compounds; C014741:bis(1,10-phenanthroline)copper(2+) ion; D004228:Dithionitrobenzoic Acid; C020582:glucosamine-6-phosphate isomerase; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C015001:arsenite; D001151:Arsenic