Purification and characterization of glucosamine-6-phosphate deaminase from dog kidney cortex.
Glucosamine-6-phosphate deaminase (EC 5.3.1.10) from dog kidney cortex was purified to homogeneity, as judged by several criteria of purity. The purification procedure was based on two biospecific affinity chromatography steps, one of them using N-epsilon-amino-n-hexanoyl-D-glucosamine-6-phosphate agarose, an immobilized analog of the allosteric ligand, and the other by binding the enzyme to phosphocellulose followed by substrate elution, which behaved as an active-site affinity chromatography. The enzyme is an hexameric protein of about 180 kDa composed of subunits of 30.4 kDa; its isoelectric point was 5.7. The sedimentation coefficient was 8.3S, and its frictional ratio was 1.28, indicating that dog deaminase is a globular protein. The enzyme displays positive homotropic cooperativity toward D-glucosamine-6-phosphate (Hill coefficient = 2.1, pH 8.8). Cooperativity was completely abolished by saturating concentrations of GlcNAc6P; this allosteric modulator activated the reaction with a typical K-effect. Under hyperbolic kinetics, a Km value of 0.25 +/- 0.02 mM for D-glucosamine-6-phosphate was obtained. Assuming six catalytic sites per molecule, kcat is 42 s-1. Substrate-velocity data were fitted to the Monod's allosteric model for the exclusive-binding case for both substrate and activator, with two interacting substrate sites. The Kdis for N-acetyl-D-glucosamine-6-phosphate was estimated at 14 microM.
Lara-Lemus
R
R
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, D.F.
Libreros-Minotta
C A
CA
Altamirano
M M
MM
Calcagno
M L
ML
D019747:Aldose-Ketose Isomerases; D000818:Animals; D002238:Carbohydrate Epimerases / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007672:Kidney Cortex / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight
D046911:Macromolecular Substances; C020582:glucosamine-6-phosphate isomerase; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases