Publisher Summary This chapter describes the assay method, purification procedure, and properties of glucosamine 6-phosphate deaminase from Escherichia coli. Crude extracts convert glucosamine 6-phosphate to a mixture of hexose phosphates which are assayed by the anthrone procedure. Purified preparations, free of phosphoglucoisomerase, yield fructose 6-phosphate, which may be determined with the Roe resoreinol reagent. The purified enzyme exhibited activity with glucosamine 6-phosphate but with none of the following: glucosamine, N-acetylglucosamine 6-phosphate, galactosamine 6-phosphate, N-acetylgalactosamine 6-phosphate, and mannosamine 6-phosphate. The purified enzyme showed no cofactor requirements after treatment with mixed-bed ion-exchange resin or after prolonged dialysis against Versene. Although the equilibrium constant for the reaction has not been determined, fructose 6-phosphate and ammonia formation are strongly favored over glucosamine 6-phosphate synthesis. The equilibrium can be shifted toward glucosamine 6-phosphate synthesis by coupling the deaminase reaction with purified glucosamine 6-phosphate N-acetylase and acetyl coenzyme A, resulting in a rapid formation of N-acetylglucosamine 6-phosphate.