Abstract
Abstract Glucosamine 6-phosphate synthesis has been examined in young adult houseflies (Musca domestica Linnaeus) at a time of active feeding and chitin production and shown to be dependent upon fructose 6-phosphate and inorganic ammonia in a reaction catalyzed by glucosamine phosphate isomerase (2-amino-2-deoxy-d-glucose 6-phosphate ketol isomerase (deaminating), EC 5.3.1.10). The 580-fold purified enzyme is specific for fructose 6-phosphate and some form of inorganic ammonia, and the product has been identified as glucosamine 6-phosphate. The molecular weight of the enzyme is about 154,000. The pH optimum of the enzyme for amination of fructose 6-phosphate is 7.1 to 7.3 in Tris-HCl and Tris-maleate buffers, between 7.7 and 8.2 in Tris-HCl containing inorganic phosphate, and 8.3 in Tris-HCl containing glucose 6-phosphate. The shift in the pH optimum in the presence of inorganic phosphate and glucose 6-phosphate accompanies the activation of the enzyme in the direction of amination by these two compounds. The pH optimum for the deamination of glucosamine 6-phosphate appears to occupy a broad region between pH 6.8 and 8.3 and is almost unaffected by either inorganic phosphate or glucose 6-phosphate, as is the rate of the reaction. The Km values for fructose 6-phosphate, ammonia, and glucosamine 6-phosphate are 36 mm, 25 mm, and 17 mm, respectively. In the presence of 5 mm glucose 6-phosphate, the Km values for fructose 6-phosphate and ammonium chloride are 3 mm and 3.3 mm, respectively, the Km for glucosamine 6-phosphate remaining unchanged. The activation constant for glucose 6-phosphate is 9.5 mm and the data indicate binding at one allosteric site only. N-Acetylglucosamine 6-phosphate activates the enzyme in both directions, the Km for glucosamine 6-phosphate being less than 0.5 mm. The equilibrium constant is in the range of 0.096 to 0.18 m for Keq = (fructose 6-phosphate) (ammonium chloride)/ (glucosamine 6-phosphate). In the presence of saturating concentrations of substrates and activators, the initial rate of amination in the presence of N-acetylglucosamine 6-phosphate is 3.8 times the rate of deamination. The initial rate of amination in the presence of glucose 6-phosphate is 3.1 times the rate of deamination in the presence of N-acetylglucosamine 6-phosphate, glucose 6-phosphate not altering the rate of deamination. Mannose 6-phosphate inhibits the amination of fructose 6-phosphate, but this is partially reversed by glucose and N-acetylglucosamine 6-phosphates. In the presence of N-acetylglucosamine 6-phosphate, glucose 6-phosphate has no effect on amination of fructose 6-phosphate. It is not clear whether glucose and N-acetylglucosamine 6-phosphates bind at the same site. Inorganic phosphate, itself an activator, partially inhibits activation by glucose 6-phosphate. Glucosamine 6-phosphate has no effect on the enzyme other than as a substrate. The absence in these animals of an enzyme which catalyzes formation of glucosamine 6-phosphate from l-glutamine and fructose 6-phosphate, the activation of the enzyme by glucose 6-phosphate only in the direction of amination of fructose 6-phosphate, and the higher initial rates of amination (3.8 times) than of deamination are evidence that the enzyme is a link in the synthetic pathway leading to chitin.
Highlights
This paper reports the purification and properties of glucosamine phosphate isomerase isolated from actively feeding adult houseflies, Xusca domestica L., at a stage in the life cycle when chitin is beirrg synthesized from neutral carbohydrates
A survey was made of extracts prepared in various ways from I- to 4-day-old adult flies in an effort to determine the relative importance of various enzyme systems in the synthesis of glucosamine B-phosphate
Extracts of fresh flies prepared in phosphate buffer, phosphate buffer containing low concentrations of n-glutamine, Tris buffer, and distilled water were tested for ability to synthesize glucosamine B-phosphate from fructose B-phosphate and the following compounds: L-glutamine, L-asparagine, L-alanine, and ammonium chloride
Summary
Enzyme Assays-For routine analysis of glucosamine 6-phosphate formation, the incubation mixture contained the following in a total volume of 1.0 ml: 50 pmoles of Tris-HCl buffer, pH 8.5; 2.5 pmoles of EDTA; 10 pmoles of fructose g-phosphate; 20 pmoles of NH&l-KOH, pH 8.5; enzyme. The mixture was kept in a water bath at 37” for the desired time, and the reaction stopped by heating in boiling water for 2 min, followed by cooling in ice water. A sample containing up to 200 nmoles in 0.8 ml was treated with 0.5 ml of 1.12 M H3B03 and 0.56 M KOH, and 0.1 ml of freshly prepared ice-cold 5 ‘% acetic anhydride in water, shaken, and incubated for a minimum of 4 min at room temperature. The tubes were placed in a boiling water bath for a minimum of 9 min, cooled in ice wat.er, and warmed to room temperature. Six milliliters of freshly prepared Ehrlich reagent
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