Effects of Temperature and Energy on Stability of Oligomeric Enzyme Probed on Electrospray Ionization Mass Spectrometry

Abstract

Escherichia coli 3-Deoxy-D-manno-octulosonate 8-phosphate(KDO8P) synthase catalyzed the condensation reaction between D-arabinose 5-phosphate(A5P) and phosphoenolpyruvate(PEP) to form KDO8P and inorganic phosphate(Pi). The noncovalent tetrameric association of KDO8P synthase was observed and dissociated in gas phase by means of electrospray ionization mass spectrometry under the very “soft” conditions. The results indicate that PEP-bound enzyme generated abundant tetrameric species as well as monomeric species at the “soft” conditions, whereas, the unbound enzyme favored the formation of a dimeric species. The mass spectra of the mixture of the enzyme with one of substrates, PEP, and A5P or one of products, KDO8P and Pi show that the complex of the unbound enzyme with PEP or Pi was prone to the formation of a monomeric species, whereas, that of the unbound enzyme with A5P or KDO8P was similar to the unbound enzyme. The intensity of the dimeric species increased with the increase of temperature at a collision voltage of 10 V. Taken together, the results presented here suggest that mass spectrometry will be a powerful tool to explore subtile conformational changes and/or subunit-subunit interactions of multiprotein assembly induced by ligand-binding and/or the changes of environmental conditions.