Probing the Oligomeric Structure of an Enzyme by Electrospray Ionization Time-Of-Flight Mass Spectrometry

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 93; no. 14; pp. 6851 - 6856
• Main Authors: Fitzgerald, Michael C., Chernushevich, Igor, Standing, Kenneth G., Whitman, Christian P., Stephen B. H. Kent
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 09-07-1996; National Acad Sciences; National Academy of Sciences
• Subjects: Amino acids; Bicarbonates; Chemistry; Circular Dichroism; Enzymes; Enzymes - chemistry; Ions; Isomerases - chemistry; Isomerases - metabolism; Kinetics; Macromolecular Substances; Mass spectra; Mass Spectrometry - methods; Mass spectroscopy; Monomers; Peptide Fragments - chemical synthesis; Peptide Fragments - chemistry; Protein Conformation; Protein Denaturation; Pseudomonas putida; Pseudomonas putida - enzymology; Quaternary ammonium compounds; Scientific imaging; Structure-Activity Relationship
• DOI: 10.1073/pnas.93.14.6851
• EISSN: 1091-6490
• ISSN: 0027-8424

Electrospray ionization time-of-flight (ESITOF) 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 ESITOF measurements were consistent with structural data obtained from circular dichroism spectroscopy. In the context of kinetic data collected for 4OT and these analogues, ESITOF mass spectrometry also provided important evidence for the structural and mechanistic significance of the catalytically important Pro1 residue in 4OT.