Pinot F, Grant D F, Beetham J K, Parker A G, Borhan B, Landt S, Jones A D, Hammock B D
Department of Entomology, University of California, Davis 95616-8584, USA.
J Biol Chem. 1995 Apr 7;270(14):7968-74. doi: 10.1074/jbc.270.14.7968.
In order to investigate the involvement of amino acids in the catalytic mechanism of the soluble epoxide hydrolase, different mutants of the murine enzyme were produced using the baculovirus expression system. Our results are consistent with the involvement of Asp-333 and His-523 in a catalytic mechanism similar to that of other alpha/beta hydrolase fold enzymes. Mutation of His-263 to asparagine led to the loss of approximately half the specific activity compared to wild-type enzyme. When His-332 was replaced by asparagine, 96.7% of the specific activity was lost and mutation of the conserved His-523 to glutamine led to a more dramatic loss of 99.9% of the specific activity. No activity was detectable after the replacement of Asp-333 by serine. However, more than 20% of the wild-type activity was retained in an Asp-333-->Asn mutant produced in Spodoptera frugiperda cells. We purified, by affinity chromatography, the wild-type and the Asp-333-->Asn mutant enzymes produced in Trichoplusia ni cells. We labeled these enzymes by incubating them with the epoxide containing radiolabeled substrate juvenile hormone III (JH III). The purified Asp-333-->Asn mutant bound 6% of the substrate compared to the wild-type soluble epoxide hydrolase. The mutant also showed 8% of the specific activity of the wild-type. Preincubation of the purified Asp-333-->Asn mutant at 37 degrees C (pH 8), however, led to a complete recovery of activity and to a change of isoelectric point (pI), both of which are consistent with hydrolysis of Asn-333 to aspartic acid. This intramolecular hydrolysis of asparagine to aspartic acid may explain the activity observed in this mutant. Wild-type enzyme that had been radiolabeled with the substrate was digested with trypsin. Using reverse phase-high pressure liquid chromatography, we isolated four radiolabeled peptides of similar polarity. These peptides were not radiolabeled if the enzyme was preincubated with a selective competitive inhibitor of soluble epoxide hydrolase 4-fluorochalcone oxide. This strongly suggested that these peptides contained a catalytic amino acid. Each peptide was characterized with N-terminal amino acid sequencing and electrospray mass spectrometry. All four radiolabeled peptides contained overlapping sequences. The only aspartic acid present in all four peptides and conserved in all epoxide hydrolases was Asp-333. These peptides resulted from cleavage at different trypsin sites and the mass of each was consistent with the covalent linkage of Asp-333 to the substrate.
为了研究氨基酸在可溶性环氧化物水解酶催化机制中的作用，利用杆状病毒表达系统制备了小鼠酶的不同突变体。我们的结果与天冬氨酸-333（Asp-333）和组氨酸-523（His-523）参与一种类似于其他α/β水解酶折叠酶的催化机制一致。将组氨酸-263突变为天冬酰胺导致与野生型酶相比，比活性丧失了约一半。当组氨酸-332被天冬酰胺取代时，96.7%的比活性丧失，而保守的组氨酸-523突变为谷氨酰胺导致比活性更显著地丧失99.9%。用丝氨酸取代天冬氨酸-333后未检测到活性。然而，在草地贪夜蛾细胞中产生的天冬氨酸-333→天冬酰胺（Asp-333-->Asn）突变体中保留了超过20%的野生型活性。我们通过亲和色谱法纯化了在粉纹夜蛾细胞中产生的野生型和天冬氨酸-333→天冬酰胺突变体酶。我们通过将它们与含有放射性标记底物保幼激素III（JH III）的环氧化物孵育来标记这些酶。与野生型可溶性环氧化物水解酶相比，纯化的天冬氨酸-333→天冬酰胺突变体结合了6%的底物。该突变体还显示出野生型比活性的8%。然而，将纯化的天冬氨酸-333→天冬酰胺突变体在37℃（pH 8）下预孵育导致活性完全恢复和等电点（pI）改变，这两者都与天冬酰胺-333水解成天冬氨酸一致。天冬酰胺向天冬氨酸的这种分子内水解可能解释了在该突变体中观察到的活性。用底物进行放射性标记的野生型酶用胰蛋白酶消化。使用反相高压液相色谱法，我们分离出了四种极性相似的放射性标记肽段。如果该酶与可溶性环氧化物水解酶的选择性竞争性抑制剂4-氟查耳酮氧化物预孵育，这些肽段不会被放射性标记。这强烈表明这些肽段含有催化氨基酸。每个肽段通过N端氨基酸测序和电喷雾质谱进行表征。所有四个放射性标记肽段都包含重叠序列。所有四个肽段中都存在且在所有环氧化物水解酶中保守的唯一天冬氨酸是天冬氨酸-333。这些肽段是在不同的胰蛋白酶位点切割产生的，每个肽段的质量与天冬氨酸-333与底物的共价连接一致。
