MOLECULAR AND BIOCHEMICAL-EVIDENCE FOR THE INVOLVEMENT OF THE ASP-333-HIS-523 PAIR IN CATALYTIC MECHANISM OF SOLUBLE EPOXIDE HYDROLASE
MOLECULAR AND BIOCHEMICAL-EVIDENCE FOR THE INVOLVEMENT OF THE ASP-333-HIS-523 PAIR IN CATALYTIC MECHANISM OF SOLUBLE EPOXIDE HYDROLASE
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DOI：
10.1074/jbc.270.14.7968
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发表时间：
1995-04-07
影响因子：
4.8
通讯作者：
HAMMOCK, BD
中科院分区：
文献类型：
--
作者：
PINOT, F;GRANT, DF;HAMMOCK, BD
为了研究氨基酸在可溶性环氧化物水解酶催化机制中的参与，使用杆状病毒表达系统产生了小鼠酶的不同突变体。我们的结果与 Asp-333 和 His-523 参与的催化机制与其他 α/β 水解酶折叠酶类似的催化机制一致。 Mutation of His-263 to asparagine led to the loss of approximately half the specific activity compared to wild-type enzyme.当 His-332 被天冬酰胺取代时，96.7% 的比活性丧失，而保守的 His-523 突变为谷氨酰胺导致 99.9% 的比活性更急剧地丧失。 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 produce din Spodoptera frugiperda cells. We purified, by affinity chromatography, the wild-type and the Asp-333 --> Asn mutant enzymes produced in Trichoplusia ni cells.我们通过将这些酶与含有放射性标记底物保幼激素 III (JH III) 的环氧化物一起孵育来标记这些酶。与野生型可溶性环氧化物水解酶相比，纯化的 Asp-333 --> Asn 突变体结合了 6% 的底物。该突变体还表现出野生型的 8% 的比活性。然而，纯化的 Asp-333 --> Asn 突变体在 37°C (pH 8) 下预孵育会导致活性完全恢复并导致等电点 (pI) 发生变化，这两者都与 Asn-333 水解为天冬氨酸一致。 This intramolecular hydrolysis of asparagine to aspartic acid may explain the activity observed in this mutant.用胰蛋白酶消化已用底物放射性标记的野生型酶。 Using reverse phase-high pressure liquid chromatography, we isolated four radiolabeled peptides of similar polarity.如果酶与可溶性环氧化物水解酶 4-氟查尔酮氧化物的选择性竞争性抑制剂预孵育，则这些肽不会被放射性标记。这强烈表明这些肽含有催化氨基酸。每种肽均通过 N 端氨基酸测序和电喷雾质谱进行表征。所有四种放射性标记的肽都包含重叠序列。 The only aspartic acid present in all four peptides and conserved in all epoxide hydrolases was Asp-333.这些肽是由不同胰蛋白酶位点裂解产生的，每种肽的质量与 Asp-333 与底物的共价连接一致。
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 produce din 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.