Quantitative evaluation of noncovalent chorismate mutase-inhibitor binding by ESI-MS.
Electrospray time-of-flight mass spectrometry was used to quantitatively determine the dissociation constant of chorismate mutase and a transition state analogue inhibitor. This system presents a fairly complex stoichiometry because the native protein is a homotrimer with three equal and independent substrate binding sites. We can detect the chorismate mutase trimer as well as chorismate mutase-inhibitor complexes by choosing appropriate conditions in the ESI source. To verify that the protein-inhibitor complexes are specific, titration experiments with different enzyme variants and different inhibitors were performed. A plot of the number of bound inhibitors versus added inhibitor concentration revealed saturation behavior with 3:1 (inhibitor:functional trimer) stoichiometry for the TSA. The soft ESI conditions, the relatively high protein mass of 43.5 kDa, and the low charge state (high m/z) result in broad peaks, a typical problem in analyzing noncovalent protein complexes. Due to the low molecular weight of the TSA (226 Da) the peaks of the free protein and the protein with one, two or three inhibitors bound cannot be clearly resolved. For data analysis, relative peak areas of the deconvoluted spectra of chorismate mutase-inhibitor complexes were obtained by fitting appropriate peak shapes to the signals corresponding to the free enzyme and its complexes with one, two, or three inhibitor molecules. From the relative peak areas we were able to calculate a dissociation constant that agreed well with known solution-phase data. This method may be generally useful for interpreting mass spectra of noncovalent complexes that exhibit broad peaks in the high m/z range.
Wendt
Silke
S
Laboratory of Organic Chemistry, Swiss Federal Institute of Technology (ETH), Zürich, Switzerland.
McCombie
Gregor
G
Daniel
Jürg
J
Kienhöfer
Alexander
A
Hilvert
Donald
D
Zenobi
Renato
R
D001412:Bacillus subtilis / Q000201:enzymology; D002826:Chorismate Mutase / Q000037:antagonists & inhibitors* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D021241:Spectrometry, Mass, Electrospray Ionization
D004791:Enzyme Inhibitors; D002826:Chorismate Mutase