Mechanism and kinetics of metalloenzyme phosphomannose isomerase: Measurement of dissociation constants and effect of zinc binding using ESI-FTICR mass spectrometry

Electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry was used to study the noncovalent complexation of a metalloenzyme, phosphomannose isomerase (PMI), which catalyzes the interconversion of mannose 6-phosphate and fructose 6-phosphate. The zinc cofactor binding effect and the noncovalent interactions of the holoenzyme with its two natural substrates and two inhibitors, erythrose 4-phosphate and mannitol 1-phosphate, were investigated. Under nondenaturing conditions, the intact zinc-containing monomeric protein ions were reproducibly observed with no dissociation. Molecular ions corresponding to apo-PMI monomer were obtained by dialyzing the holoenzyme against EDTA. The binding/release of the metal ion did not alter the charge-state distributions of the protein to any significant extent, but changed the binding affinity of the substrates by at least 5-fold. Using ESI-FTICR mass spectrometry, the binding stoichiometry and specificity of the enzyme-substrate and enzyme-inhibitor complexes were directly determined. The first time report of the apparent dissociation constant for the isomeric substrates of PMI was measured to be 88.8 μM. The relative dissociation constant of the two inhibitors derived from gas-phase noncovalent complexation was very similar to the relative inhibition constant derived from solution phase kinetics.