Analysis of the Escherichia coli glucosamine-6-phosphate synthase activity by isothermal titration calorimetry and differential scanning calorimetry.
Glucosamine-6-phosphate synthase (GlmS) is responsible for the first and rate-limiting step in the hexosamine biosynthetic pathway. It catalyzes the conversion of D-fructose-6P (F6P) into D-glucosamine-6P (GlcN6P) using L-glutamine (Gln) as nitrogen donor (synthase activity) according to an ordered bi-bi process where F6P binds first. In the absence of F6P, the enzyme exhibits a weak hydrolyzing activity of Gln into Glu and ammonia (glutaminase activity), whereas the presence of F6P strongly stimulates it (hemi-synthase activity). Until now, these different activities were indirectly measured using either coupled enzyme or colorimetric methods. In this work, we have developed a direct assay monitoring the heat released by the reaction. Isothermal titration calorimetry and differential scanning calorimetry were used to determine kinetic and thermodynamic parameters of GlmS. The direct determination at 37 degrees C of kinetic parameters and affinity constants for both F6P and Gln demonstrated that part of the ammonia produced by Gln hydrolysis in the presence of both substrates is not used for the formation of the GlcN6P. The full characterization of this phenomenon allowed to identify experimental conditions where this leak of ammonia is negligible. Enthalpy measurements at 25 degrees C in buffers of various heats of protonation demonstrated that no proton exchange with the medium occurred during the enzyme-catalyzed glutaminase or synthase reaction suggesting for the first time that both products are released as a globally neutral pair composed by the Glu carboxylic side chain and the GlcN6P amine function. Finally we showed that the oligomerization state of GlmS is concentration-dependent.
Valerio-Lepiniec
Marie
M
Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619, Université de Paris-Sud 11, Orsay, France. marielle.valerio@u-psud.fr
Aumont-Nicaise
Magali
M
Roux
Céline
C
Raynal
Bertrand
B
England
Patrick
P
Badet
Bernard
B
Badet-Denisot
Marie-Ange
MA
Desmadril
Michel
M
D002152:Calorimetry, Differential Scanning; D002153:Calorimetry, Indirect; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005944:Glucosamine / Q000031:analogs & derivatives / Q000737:chemistry; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives / Q000737:chemistry; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000737:chemistry*; D006358:Hot Temperature; D007700:Kinetics
D029968:Escherichia coli Proteins; C001293:glucosamine 6-phosphate; D019298:Glucose-6-Phosphate; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D005944:Glucosamine