The product of the natural reaction catalyzed by 4-oxalocrotonate tautomerase becomes an affinity label of its mutant

Ashraf Brik, Philip E. Dawson, Ehud Keinan

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

4-Oxalocrotonate tautomerase (4-OT) catalyzes the isomerization of 4-oxalocrotonate, 1, to 2-oxo-3E-hexenedioate, 3, using a general acid/base mechanism that involves a conserved N-terminal proline residue. The P1A and P1G mutants have been shown to catalyze this isomerization but at reduced rates. Analysis of these mutants by mass spectrometry demonstrated that P1A is susceptible to a 1,4-addition of the N-terminal primary amine across the double bond of enone 3 to form a covalent adduct. Although slower than the isomerization reaction, the addition is fast, with 50% of the active sites being alkylated within 12 min. By contrast, the wt4-OT shows no detectable modification over 24 h. These results support the hypothesis that avoidance of nucleophilic reactions, such as the irreversible Michael addition to the product, could be a contributing factor in the evolutionary conservation of N-terminal proline residues in 4OT.

Original languageEnglish
Pages (from-to)3891-3897
Number of pages7
JournalBioorganic and Medicinal Chemistry
Volume10
Issue number12
DOIs
StatePublished - 1 Dec 2002
Externally publishedYes

Fingerprint

Dive into the research topics of 'The product of the natural reaction catalyzed by 4-oxalocrotonate tautomerase becomes an affinity label of its mutant'. Together they form a unique fingerprint.

Cite this