Brik Ashraf, Dawson Philip E, Keinan Ehud
Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Bioorg Med Chem. 2002 Dec;10(12):3891-7. doi: 10.1016/s0968-0896(02)00385-1.
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.
4-草酰巴豆酸互变异构酶（4-OT）催化4-草酰巴豆酸（1）异构化为2-氧代-3E-己烯二酸（3），其采用涉及保守N端脯氨酸残基的一般酸碱机制。已证明P1A和P1G突变体可催化这种异构化，但速率降低。通过质谱分析这些突变体表明，P1A易受N端伯胺在烯酮3的双键上进行1,4-加成以形成共价加合物的影响。尽管比异构化反应慢，但加成反应很快，12分钟内50%的活性位点被烷基化。相比之下，野生型4-OT在24小时内未显示出可检测到的修饰。这些结果支持这样一种假设，即避免亲核反应，如对产物的不可逆迈克尔加成，可能是4OT中N端脯氨酸残基进化保守性的一个促成因素。
