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 tautomeras e (EC 5.3.2; 4OT), and four analogues prepared by total chemical synth esis, Wild-type 4OT is a hexamer of 62 amino acid subunits and contain s no cysteine residues. The analogues were: (desPro(1))4OT, a truncate d construct in which Pro(1) was deleted; (Cpc(1))40T in which Pro(1) w as replaced with cyclopentane carboxylate; a derivative [Met(O)(45)]4O T 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 n ative enzyme was fully active (5 mM ammonium bicarbonate buffer, pH 7. 5) gave the intact hexamer as the major species detected by TOF mass s pectrometry, In contrast, analysis of [Met(O)(45)]4OT and (desPro(1))4 OT under similar conditions yielded predominantly monomer ions. The ES I-TOF measurements were consistent with structural data obtained from circular dichroism spectroscopy. In the context of kinetic data collec ted for 4OT and these analogues, ESI-TOF mass spectrometry also provid ed important evidence for the structural and mechanistic significance of the catalytically important Pro(1) residue in 4OT.