Williams T A, Michaud A, Houard X, Chauvet M T, Soubrier F, Corvol P
INSERM U36, Collège de France, Paris, France.
Biochem J. 1996 Aug 15;318 ( Pt 1)(Pt 1):125-31. doi: 10.1042/bj3180125.
Drosophila melanogaster angiotensin I-converting enzyme (AnCE) is a secreted single-domain homologue of mammalian angiotensin I-converting enzyme (ACE) which comprises two domains (N and C domains). In order to characterize in detail the enzymic properties of AnCE and to study the influence of glycosylation on the secretion and enzymic activity of this enzyme, we overexpressed AnCE (expression level, 160 mg/l) and an unglycosylated mutant (expression level, 43 mg/l) in the yeast Pichia pastoris. The recombinant enzyme was apparently homogeneous on SDS/PAGE without purification and partial deglycosylation demonstrated that all three potential sites for N-linked glycosylation were occupied by oligosaccharide chains. Each N-glycosylation sequence (Asn-Xaa-Ser/Thr) was disrupted by substituting a glutamine for the asparagine residue at amino acid positions 53, 196 and 311 by site-directed mutagenesis to produce a single mutant. Expression of the unglycosylated mutant in Pichia produced a secreted catalytically active enzyme (AnCE delta CHO). This mutant displayed unaltered kinetics for the hydrolyses of hippuryl-His-Leu, angiotensin 1 and N-acetyl-Ser-Asp-Lys-Pro (AcSDKP) and was equally sensitive to ACE inhibitors compared with wild-type AnCE. However, AnCE delta CHO was less stable, displaying a half-life of 4.94 h at 37 degrees C, compared with AnCE which retained full activity under the same conditions. Two catalytic criteria demonstrate the functional resemblance of AnCE with the human ACE C domain: first, the kcat/Km of AcSDKP hydrolysis and secondly, the kcat/Km and optimal chloride concentration for hippuryl-His-Leu hydrolysis. A range of ACE inhibitors were far less potent towards AnCE compared with the human ACE domains, except for captopril which suggests an alternative structure in AnCE corresponding to the region of the S1 subsite in the human ACE active sites.
果蝇血管紧张素I转换酶（AnCE）是哺乳动物血管紧张素I转换酶（ACE）的一种分泌型单结构域同源物，后者由两个结构域（N结构域和C结构域）组成。为了详细表征AnCE的酶学特性，并研究糖基化对该酶分泌和酶活性的影响，我们在毕赤酵母中过表达了AnCE（表达水平为160 mg/l）和一个非糖基化突变体（表达水平为43 mg/l）。重组酶在未经纯化的SDS/PAGE上显然是均一的，部分去糖基化表明所有三个潜在的N-糖基化位点都被寡糖链占据。通过定点诱变将谷氨酰胺取代氨基酸位置53、196和311处的天冬酰胺残基，破坏了每个N-糖基化序列（Asn-Xaa-Ser/Thr），从而产生一个单突变体。非糖基化突变体在毕赤酵母中的表达产生了一种分泌型催化活性酶（AnCE delta CHO）。该突变体对马尿酰-His-Leu、血管紧张素1和N-乙酰-Ser-Asp-Lys-Pro（AcSDKP）水解的动力学没有改变，与野生型AnCE相比，对ACE抑制剂同样敏感。然而，AnCE delta CHO稳定性较差，在37℃下的半衰期为4.94小时，而AnCE在相同条件下保留了全部活性。两个催化标准证明了AnCE与人类ACE C结构域的功能相似性：第一，AcSDKP水解的kcat/Km；第二，马尿酰-His-Leu水解的kcat/Km和最佳氯化物浓度。与人类ACE结构域相比，一系列ACE抑制剂对AnCE的效力要低得多，除了卡托普利，这表明AnCE中对应于人类ACE活性位点S1亚位点区域的结构有所不同。
