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: (desPro1)4OT, a truncated construct in which Pro1 was deleted; (Cpc1)4OT in which Pro1 was replaced with cyclopentane carboxylate; a derivative [Met(O)45]4OT in which Met45 was oxidized to the sulfoxide; and an analogue (Nle45)4OT in which Met45 was replaced with norleucine. ESI of (Nle45)4OT, (Cpc1)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 (desPro1)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 Pro1 residue in 4OT.
Fitzgerald
M C
MC
The Scripps Research Institute, La Jolla, CA 92037, USA.
Chernushevich
I
I
Standing
K G
KG
Whitman
C P
CP
Kent
S B
SB
D002942:Circular Dichroism; D004798:Enzymes / Q000737:chemistry*; D007535:Isomerases / Q000737:chemistry* / Q000378:metabolism; D046911:Macromolecular Substances; D013058:Mass Spectrometry / Q000379:methods*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D016958:Pseudomonas putida / Q000201:enzymology; D013329:Structure-Activity Relationship
D004798:Enzymes; D046911:Macromolecular Substances; D010446:Peptide Fragments; C021918:4-oxalocrotonate tautomerase; D007535:Isomerases