Purification, molecular and kinetic properties of glucosamine-6-phosphate isomerase (deaminase) from Escherichia coli.
Glucosamine-6-phosphate isomerase (deaminase), (2-amino-2-deoxy-D-glucose-6-phosphate ketol isomerase (deaminating), EC 5.3.1.10) has been purified to homogeneity from Escherichia coli B as judged by several criteria of purity. The procedure included ammonium sulfate fractionation, anion-exchange chromatography and a biospecific affinity chromatography step with N-epsilon-amino-n- caproyl -D-glucosamine 6-phosphate bound to agarose as the ligand, the elution being performed with GlcNAc6 P. The enzyme appears to be an hexamer of about 178 kDa, composed of six subunits of 29 700 +/- 300 Da; the isoelectric point was 6.0-6.1 and the sedimentation constant 9.0 S. The amino-acid composition of the enzyme was determined and a value for E1%275 of 4.55 was calculated. The molecular activity was 1800 s-1 for the deamination reaction and 455 s-1 for the reaction of GlcN6 P formation. A positive homotropic cooperativity was found for both sugar substrates; it was stronger for GlcN6 P in the deamination reaction (Hill number 2.7 at pH 7.7). Ammonia behaved as a Michaelian substrate. Cooperativity was abolished by 0.1 mM GlcNAc6 P; this allosteric modulator activated the reaction in both directions, with a positive K-effect upon both sugar phosphates, but had no effect on Km for ammonia. The initial velocity patterns for the amination reaction were obtained under conditions of hyperbolic kinetics produced by GlcNAc6 P; the Km values for the allosteric substrates were determined under the same conditions, and their dependence upon pH was studied.
D019747:Aldose-Ketose Isomerases; D000494:Allosteric Regulation; D002238:Carbohydrate Epimerases / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight
D046911:Macromolecular Substances; C020582:glucosamine-6-phosphate isomerase; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases