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

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 PIA and PIG mutants have been shown to catalyze this isomerization but at reduced rates. Analysis of these mutants by mass spectrometry demonstrated that PIA is susceptible to a 1.4-addition of the N-terininal 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 shoals no detectable modification over 24h. 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 40T. (C) 2002 Elsevier Science Ltd. All rights reserved.