TY - JOUR
T1 - The product of the natural reaction catalyzed by 4-oxalocrotonate tautomerase becomes an affinity label of its mutant
AU - Brik, Ashraf
AU - Dawson, Philip E.
AU - Keinan, Ehud
N1 - Funding Information:
This work was supported by the Skaggs Institute for Chemical Biology, (P.D. and E.K.), NIH (GM59380) (P.D.), and the Israel Ministry of Science (A.B.). E.K. is the incumbent of the Benno Gitter & Ilana Ben-Ami Chair of Biotechnology, Technion. We thank Dr. Steven Bark of TSRI open access mass spectrometry facilities for help with the MS/MS experiments.
PY - 2002/12/1
Y1 - 2002/12/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0036973289&partnerID=8YFLogxK
U2 - 10.1016/S0968-0896(02)00385-1
DO - 10.1016/S0968-0896(02)00385-1
M3 - Article
AN - SCOPUS:0036973289
SN - 0968-0896
VL - 10
SP - 3891
EP - 3897
JO - Bioorganic and Medicinal Chemistry
JF - Bioorganic and Medicinal Chemistry
IS - 12
ER -