title: Monitoring the dynamics of monomer exchange using electrospray mass spectrometry: the case of the dimeric glucosamine-6-phosphate synthase.
abstract: Escherichia coli glucosamine-6-phosphate synthase (GlmS) is a dimeric enzyme from the glutamine-dependent amidotransferases family, which catalyses the conversion of D-fructose-6-phosphate (Fru6P) and glutamine (Gln) into D-glucosamine-6-phosphate (GlcN6P) and glutamate, respectively. Extensive X-ray crystallography investigations have been reported, highlighting the importance of the dimeric association to form the sugar active site as well as significant conformational changes of the protein upon substrate and product binding. In the present work, an approach based on time-resolved noncovalent mass spectrometry has been developed to study the dynamics of GlmS subunit exchange. Using (14)N versus (15)N labeled proteins, the kinetics of GlmS subunit exchange was monitored with the wild-type enzyme in the presence of different substrates and products as well as with the protein bearing a key amino acid mutation specially designed to weaken the dimer interface. Determination of rate constants of subunit exchange revealed important modifications of the protein dynamics: while glutamine, glutamate, and K603A mutation accelerates subunit exchange, Fru6P and GlcN6P totally prevent it. These results are described in light of the available structural information, providing additional useful data for both the characterization of GlmS catalytic process and the design of new GlmS inhibitors. Finally, time-resolved noncovalent MS can be proposed as an additional biophysical technique for real-time monitoring of protein dynamics.
authors.affiliation: Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), Université de Strasbourg, IPHC, 25 rue Becquerel 67087, Strasbourg, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Chevreux
authors.forename: Cédric
authors.initials: C
authors.lastname: Atmanene
authors.forename: Philippe
authors.initials: P
authors.lastname: Lopez
authors.forename: Jamal
authors.initials: J
authors.lastname: Ouazzani
authors.forename: Alain
authors.initials: A
authors.lastname: Van Dorsselaer
authors.forename: Bernard
authors.initials: B
authors.lastname: Badet
authors.forename: Marie-Ange
authors.initials: MA
authors.lastname: Badet-Denisot
authors.forename: Sarah
authors.initials: S
authors.lastname: Sanglier-Cianférani
mesh_terms: D019943:Amino Acid Substitution; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D005973:Glutamine / Q000737:chemistry / Q000378:metabolism; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000737:chemistry* / Q000378:metabolism; D009587:Nitrogen Isotopes; D055503:Protein Multimerization; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D029968:Escherichia coli Proteins; D009587:Nitrogen Isotopes; D005973:Glutamine; D018698:Glutamic Acid; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)