Purification and some kinetic properties of rat liver glucosamine synthetase.
1. Glucosamine synthetase (l-glutamine-d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) was purified about 300-fold from rat liver by two techniques. One procedure utilized the protective action of fructose 6-phosphate and gave a relatively stable preparation, the other yielded an unstable enzyme (half-life of about 20h), free of contaminant activities, on which kinetic experiments were performed. Although the properties of the two preparations showed slight differences, the unstabilized form could be converted into the stabilized form. 2. During preparation the enzyme retained its sensitivity to the feedback inhibitor, UDP-N-acetylglucosamine. 3. The reversibility of the enzyme-catalysed reaction could not be demonstrated. There was no apparent requirement for a cofactor. 4. The pH optimum was at 7.5, at which pH the reaction obeyed a Ping Pong Bi Bi rate equation. At pH values outside the range 6.9-7.6 and at temperatures below 29 degrees C the velocity was described by an ordered Bi Bi rate equation. 5. The molecular weight of the enzyme, determined by two procedures, was 360000-400000. 6. The aminotransferase was unable to utilize ammonia as a substrate.
D000217:Acyltransferases / Q000032:analysis; D000641:Ammonia; D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D005260:Female; D005956:Glucose-6-Phosphate Isomerase / Q000032:analysis; D005972:Glutaminase / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D006882:Hydroxyapatites; D007535:Isomerases / Q000032:analysis; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008722:Methods; D008970:Molecular Weight; D010732:Phosphofructokinase-1 / Q000032:analysis; D011506:Proteins / Q000032:analysis; D051381:Rats; D013696:Temperature; D000637:Transaminases / Q000302:isolation & purification*
D006882:Hydroxyapatites; D011506:Proteins; D000641:Ammonia; D000217:Acyltransferases; D000637:Transaminases; D010732:Phosphofructokinase-1; D005972:Glutaminase; D007535:Isomerases; D005956:Glucose-6-Phosphate Isomerase