VOL. 12 (1953) BIOSYNTHESIS OF GLUCOSAMINE 21 



The result of the action of this set of enzymes is that starting with free glucosamine or 

 with glucosamine-6-phosphate, the reaction products are similar. Most of the acetyl- 

 glucosamine appears free and a part precipitates with zinc sulphate-barium hydroxide 

 as would acetylglucosamine phosphate (Table VI) . If acetylglucosamine phosphate was 

 formed, it could not have arisen by phosphorylation of acetylglucosamine, since this 

 process is not catalyzed by the extracts (Table V). Therefore, it seems logical to conclude 

 that glucosamine-6-phosphate can be acetylated directly to acetylglucosamine phosphate. 

 Table VII shows the results of the action of the acetylating system on the 

 "glucosamine" ester formed from hexose phosphate and glutamine. This substance gave 

 rise to acetylglucosamine, as did glucosamine or glucosamine-6-phosphate. 



DISCUSSION 



The substance formed from hexose-6-phosphate and glutamine gives the Elson 

 AND Morgan and Dische^^ reactions for hexosamines. It can be prepared as the barium 

 salt admixed with hexosemonophosphates. It behaves like glucosamine-6-phosphate 

 when incubated with the acetylating system of Neurospora, and after dephosphorylation 

 with phosphatase glucosamine can be identified by paper chromatography with selected 

 solvents. 



All this is considered as evidence proving that the product is glucosamine phosphate. 

 The ester gives positive reactions for hexosamines, so that a i-ester can be excluded, and 

 since a migration of the phosphate during the reaction appears unlikely, the product 

 should be glucosamine-6-phosphate. 



No evidence for the necessity of a cof actor for the formation of glucosamine 

 phosphate was obtained. Thus, no stimulation was obtained by the addition of different 

 ions, ATP, pyridoxal phosphate, UDPAG, etc. The mechanism of the reaction cannot be 

 discussed until it is decided whether the reactant is fructose-6 or glucose-6-phosphate. 

 In order to settle this point it will be necessary to obtain enzyme preparations free from 

 isomerase. 



Further investigation will also be necessary in order to decide whether the acety- 

 lation step takes place on free glucosamine, on glucosamine-6-phosphate or on both. 

 In connection with this point, it may be mentioned that Chou and Soodak^^ extracted 

 an enzyme from pigeon liver which catalyzed the acetylation of free glucosamine and 

 galactosamine, but that the corresponding phosphates were not tested. 



SUMMARY 



A partially purified enzyme has been prepared from Neurospora crassa which catalyzes the 

 formation of glucosamine phosphate from hexose-6-phosphate and glutamine. The glucosamine 

 phosphate was identified by colour reactions, by dephosphorylation and paper chromatography and 

 by its behaviour towards an acetylating system. 



Quantitative analysis of amide nitrogen, glutamate, and hexosamine agreed with the following 

 equation : 



Hexose-6-phosphate -\- glutamine > glucosamine-6-phosphate -{- glutamate 



Crude Neurospora extracts were found to phosphorylate glucosamine in the presence of ATP 

 and, when suitably supplemented, to acetylate glucosamine or glucosamine phosphate. 



References p. 2.2, 



