Borhan B, Jones A D, Pinot F, Grant D F, Kurth M J, Hammock B D
Department of Chemistry, University of California, Davis 95616, USA.
J Biol Chem. 1995 Nov 10;270(45):26923-30. doi: 10.1074/jbc.270.45.26923.
18O-Labeled epoxides of trans-1,3-diphenylpropene oxide (tDPPO) and cis-9,10-epoxystearic acid were synthesized and used to determine the regioselectivity of sEH. The nucleophilic nature of sEH catalysis was demonstrated by comparing the enzymatic and nonenzymatic hydrolysis products of tDPPO. The results from single turnover experiments with greater or equal molar equivalents of sEH:substrate were consistent with the existence of a stable intermediate formed by a nucleophilic amino acid attacking the epoxide group. Tryptic digestion of sEH previously subjected to multiple turnovers with tDPPO in H2 18O resulted in the isolation and purification of a tryptic fragment containing Asp-333. Electrospray mass spectrometry of this fragment conclusively illustrated the incorporation of 180. After complete digestion of the latter peptide it was shown that Asp-333 of sEH exhibited an increased mass. The attack by Asp-333 initiates enzymatic activity, leading to the formation of an alpha-hydroxyacyl-enzyme intermediate. Hydrolysis of the acyl-enzyme occurs by the addition of an activated water to the carbonyl carbon of the ester bond, after which the resultant tetrahedral intermediate collapses, yielding the active enzyme and the diol product.
合成了反式-1,3-二苯基环氧丙烷（tDPPO）和顺式-9,10-环氧硬脂酸的18O标记的环氧化物，并用于确定可溶性环氧化物水解酶（sEH）的区域选择性。通过比较tDPPO的酶促水解产物和非酶促水解产物，证明了sEH催化的亲核性质。用摩尔当量大于或等于sEH:底物的单次周转实验结果与亲核氨基酸攻击环氧基团形成稳定中间体的存在一致。在H2 18O中用tDPPO对sEH进行多次周转后进行胰蛋白酶消化，得到了含有天冬氨酸-333的胰蛋白酶片段的分离和纯化。该片段的电喷雾质谱最终证实了18O的掺入。在对后一种肽进行完全消化后，表明sEH的天冬氨酸-333质量增加。天冬氨酸-333的攻击引发酶活性，导致α-羟基酰基-酶中间体的形成。酰基-酶的水解是通过将活化水加到酯键的羰基碳上进行的，之后生成的四面体中间体崩溃，产生活性酶和二醇产物。
