Probing the oligomeric structure of an enzyme by electrospray ionization time-of-flight mass spectrometry

Electrospray ionization time-of-flight (ESI-TOF) mass spectrometry was used to study the quaternary structure of 4-oxalocrotonate tautomerase (EC 5.3.2; 4OT), and four analogues prepared by total chemical synthesis, Wild-type 4OT is a hexamer of 62 amino acid subunits and contains no cysteine residues. The analogues were: (desPro(1))4OT, a truncated construct in which Pro(1) was deleted; (Cpc(1))40T in which Pro(1) was replaced with cyclopentane carboxylate; a derivative [Met(O)(45)]4OT in which Met(45) was oxidized to the sulfoxide; and an analogue (Nle(45))4OT in which Met(45) was replaced with norleucine. ESI of (Nle(45))4OT, (Cpc(1))4OT, and 4OT from solution conditions under which the native enzyme was fully active (5 mM ammonium bicarbonate buffer, pH 7.5) gave the intact hexamer as the major species detected by TOF mass spectrometry, In contrast, analysis of [Met(O)(45)]4OT and (desPro(1))4OT under similar conditions yielded predominantly monomer ions. The ESI-TOF measurements were consistent with structural data obtained from circular dichroism spectroscopy. In the context of kinetic data collected for 4OT and these analogues, ESI-TOF mass spectrometry also provided important evidence for the structural and mechanistic significance of the catalytically important Pro(1) residue in 4OT.